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WO2010061347A1 - Pcr based diagnostic method for the identification of clinically important candida species - Google Patents

Pcr based diagnostic method for the identification of clinically important candida species Download PDF

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Publication number
WO2010061347A1
WO2010061347A1 PCT/IB2009/055361 IB2009055361W WO2010061347A1 WO 2010061347 A1 WO2010061347 A1 WO 2010061347A1 IB 2009055361 W IB2009055361 W IB 2009055361W WO 2010061347 A1 WO2010061347 A1 WO 2010061347A1
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seq
sequences
candida species
candida
pcr
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French (fr)
Inventor
Csaba VÁGVÖLGYI
Sándor KOCSUBÉ
Tamás PAPP
Gyula Beszedics
Ákos BESZEDICS
Lajos Sándor SZOLNOKI
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SZERODIAGNOSZTIKUM Ltd
University of Szeged
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SZERODIAGNOSZTIKUM Ltd
University of Szeged
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays

Definitions

  • the present invention is concerned with the field of PCR based diagnostic methods, more specifically with the diagnosis of infectious diseases caused by different Candida species. Still more specifically the invention provides PCR based diagnostic methods for the detection of Candida species most commonly causing invasive candidiasis.
  • the methods of the invention advantageously enable the detection of the Candida species of interest directly from heparin containing blood samples and the specific PCR primers provided by the invention also enable the detection of more than one pathogenic Candida species in one single PCR reaction based on the significantly different sizes of the species specific amplicons produced.
  • Candida albicans is the most common Candida species encountered as the causal agent of human infections, other Candida species have also been increasingly associated with disseminated disease since the 1990s.
  • Candida The genus Candida includes around 154 species. Among these, six are most frequently isolated in the case of human infections. While Candida albicans is the most abundant and significant species, Candida tropicalis, Candida glabrata, Candida parapsilosis, Candida krusei, and Candida lusitaniae are also isolated as causative agents of Candida infections. Importantly, there has been a recent increase in infections due to non -albicans Candida spp., such as Candida glabrata and Candida krusei.
  • Candida albicans (frequency: 50%)
  • Candida tropicalis (15-30%)
  • Candida glabrata (15-30%)
  • Candida parapsilosis (15-30%)
  • Candida krusei (1%)
  • Candida lusitaniae -1%)
  • C. parapsilosis is the second most common yeast species isolated from blood stream infections as reported in several surveys (Safdar et al., 2002; Messer et al., 2006; Bassetti et al., 2006).
  • Candida krusei is inherently resistant to fluconazole and is emerging as a frequent causal agent of fungemia in patients with hematologic malignant neoplasms. In neutropenic patients, (Abbas et al. 2000) C. krusei fungemia results in high mortality.
  • Candida glabrata was considered as a relatively non-pathogenic commensal fungal organism of human mucosal tissues (Fidel et al.).
  • mucosal and systemic infections caused by C. glabrata have increased significantly, especially in the human immunodefi- ciency virus-infected population.
  • a major obstacle in C. glabrata infections is their innate resistance to azole an- timycotic therapy, which is very effective in treating infections caused by other Candida species.
  • Candida gla- brata formerly known as Torulopsis glabrata, contrasts with other Candida species in its nondimorphic blasto- conidial morphology and haploid genome.
  • Candida tropicalis is a major cause of septicemia and disseminated candidiasis, especially in patients with lymphoma, leukemia and diabetes. It is the second most frequently encountered medical pathogen, next to C. albicans, and is also found as part of the normal human mucocutaneous flora. Sucrose negative variants of C. tropicalis have also been increasingly found in cases of disseminated candidiasis. Environmental isolations have been made from faeces, shrimp, kefir, and soil.
  • Candida lusitaniae is known for amphotericin B resistance (Peyron et al. 2000). The first report of fatal infection occurred in a neutropenic patient where the initial isolate was susceptible to amphotericin B but subsequent isolates had developed amphotericin B resistance. It is well-documented that this species is able to develop resistance, but some strains display resistance prior to amphotericin B therapy. Although amphotericin B resistance is a concern, reports exist where amphotericin B has been used to successfully treat fungemia in severely immunocompromised patients.
  • Candida guilliermondii Although a rare cause of invasive candidiasis, Candida guilliermondii has been reported to exhibit decreased susceptibility to antifungal agents (Girmenia et al. 2006). Aside from case reports and small surveys, there is little information regarding the epidemiology and antifungal susceptibility profile of C. guilliermondii.
  • Candida guilliermondii is infrequently isolated from blood cultures (1 to 5%), and infections caused by this spe- cies have been reported in cancer, surgical, and intensive care unit patients, including a pseudo-outbreak of can- didemia in a neonatal intensive care unit. Malignancy, neutropenia, and bone marrow transplantation have been reported as risk factors for acquiring C. guilliermondii infections.
  • Candida kefyr is a rare cause of candidiasis and is usually associated with superficial cutaneous manifestations rather than systemic disease. It has been isolated from nails and lung infections. Environmental isolations have been made from cheese and dairy products. C. kefyr infections reflect the increase in Candida species as responsible of disseminated infections in immunodepressed patients.
  • C. dubliniensis a recently described species closely related to C. albicans, has been implicated as an agent of oral candidiasis in HIV-positive persons but has also been recovered from HIV-negative persons with clinical signs of oral candidiasis and from the genital tract of some women with vaginitis. First isolated from AIDS patients in Dublin, Ireland, C. dubliniensis has a worldwide distribution.
  • ELISA techniques available for routine diagnostics are not selective enough, and are restricted mainly to C. albicans.
  • the object of the present invention is therefore to provide a reliable, rapid, cost effective and complex diag- nostic method and reagent kit suitable for the identification of all the different Candida species so far identified as significant causative agents of invasive candidiasis.
  • a further object of this invention is to provide a simple user friendly clinically applicable PCR based method and reagent kit for the detection of all clinically relevant pathogenic Candida species directly using clinical blood samples as PCR templates.
  • the present invention provides a rapid and cost effective method for identifying nine different Candida species from complex clinical samples, using a unique species specific PCR reaction.
  • the samples can be derived from any solid state media, epithelial or nail scrapings, excrement, mucosal samples or cultivated blood samples.
  • the present invention provides a PCR based method for detecting the presence of at least one pathogenic Candida species present in a heparin containing blood sample, wherein a part of said heparin containing blood sample, comprising material serving as PCR template, is directly added to a Candida species specific PCR reaction mixture.
  • the method of the invention is advantageously performed using a non-heparin resistant Taq DNA polymerase.
  • the expression "non-heparin resistant Taq DNA polymerase" in the context of the present in- vention is used to define any commercially available Taq DNA polymerase that is not commercialized or described in the art as being specially heparin resistant.
  • the blood sample used in the method of the invention is preincubated, advantageously for 1 -3 days, in conditions facilitating the growth of said at least one pathogenic Candida species being supposedly present in said blood sample, in order to en- hance the sensitivity of the method of the invention.
  • Said preincubation is advantageously done in the presence of at least one antibiotic, advantageously Neomycin and/or Chloramphenicol.
  • the PCR reaction mixture in accordance with the present invention advantageously contains less than 0.06 IU heparin, more advantageously less than 0.05 IU and a magnesium ion concentration of at least 4 mM, advantageously at least 5 mM.
  • the method of the invention is advantageously useful for the detection of pathogenic Candida species selected from the group consisting of C. glabrata, C. lusitaniae, C. guilliermondii, C. krusei, C. albicans, C. dubli- niensis, C. tropicalis, C. parapsilosis and C. kefyr.
  • the specific primer pairs of the present invention are designed in a way that the pathogenic Candida species to be identified are possible to identify on the basis of the size of the amplicon produced in the species specific PCR reaction mixture.
  • the specifically designed primer pairs used in the present invention are designed to be capable of specifically hybridizing either to a sequence re- gion present in the internal transcribed spacer (ITS) region of the ribosomal gene repeat or to a species specific sequence region present in the Phospholipase-D gene of said at least one pathogenic Candida species, which gene was surprisingly identified by the present inventors as being useful for the species specific identification of all so far examined pathogenic Candida species of interest.
  • ITS internal transcribed spacer
  • Candida species specific PCR primer pairs applied are selected from the group consisting of sequences identified by any of SEQ ID NOs from 1 to 22, sequences at least 90% identical with sequences identified by any of SEQ ID NOs from 1 to 22, and complementary sequences thereof.
  • the Candida species specific PCR reaction mixture comprises more than one, advantageously more than two PCR primer pairs being specific for more than one, advantageously more than two Candida species, and wherein said primers, upon suitable thermal cycling, enable the production of more than one, advantageously more than two species specific amplicons each having a characteristic individual size being gel-electrophoretically distinguishable from any other Candida species specific amplicon that may be produced in said Candida species specific PCR reaction mixture.
  • some of the individual PCR primers being present in said Candida species specific PCR reaction mixture are designed to be capable of specifically hybridizing to target sequences originating from more than one, advantageously more than two different Candida species being supposedly present in said blood or other biological sample, reducing thereby the overall number of different specific primers needed in the Candida species specific PCR reactions of the invention, where more than one pathogenic Candida species is identified within one single specific PCR reaction.
  • the presence of two pathogenic Candida species selected from the group consisting of C. glabrata, C. lusitaniae, C. guilliermondii, C. krusei, C. albicans, C. dubliniensis, C. tropicalis, C. parapsilosis and C. kefyr is detected in one single Candida species specific PCR reaction, wherein said primers applied in said one single Candida species specific PCR reaction are having the sequences identified by a) SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 4 for C. glabrata and C.
  • SEQ ID NO: 1 SEQ ID NO: 3 and SEQ ID NO: 5 for C. guilliermondii and C. krusei; or c) SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 9 for C. albicans and C. dubliniensis; or d) SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10 for C. tropicalis and C. parapsilosis; or e) SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13 for C. parapsilosis and C. krusei; or f) SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 for C.
  • the present inventors have surprisingly found for the first time that specific sequences present in the Phospholipase-D gene of different pathogenic Candida species of interest enable the designing of species specific primer pairs hybridizing to said species specific sequence regions and capable of giving rise thereby to species specific PCR amplicons the size of which being characteristic to said species of interest.
  • the present invention therefore, further provides a PCR based method for detecting the presence of at least one pathogenic Candida species present in a biological sample, wherein the Candida species specific PCR pri- mer pair(s) applied is(are) designed to be capable of specifically hybridizing to a species specific sequence region present in the Phospholipase-D gene of said at least one pathogenic Candida species.
  • the sequences of said Phospholipase-D specific primers of the invention are advantageously selected from a group consisting of sequences identified by any of SEQ ID NOs from 11 to 22, sequences at least 90% identical with sequences identified by any of SEQ ID NOs from 11 to 22, and complementary sequences thereof.
  • PCR based method for detecting the presence of a microorganism is used herein to identify any method that comprises at least one step wherein a specific nucleic acid sequence present in a target microorganism is amplified by at least one PCR reaction, and then said amplified specific sequence is identified.
  • the invention further provides reagent kits for detecting the presence of at least one pathogenic Candida species selected from the group consisting of C. glabrata, C. lusitaniae, C. guilliermondii, C. krusei, C. albicans, C. dubliniensis, C. tropicalis, C. parapsilosis and C. kefyr by PCR reaction, comprising at least one primer pair wherein each primer having a sequence selected from the group consisting of sequences identified by any of SEQ ID NOs from 1 to 22, sequences being at least 90% identical with sequences identified by any of SEQ ID NOs from 1 to 22, and complementary sequences thereof and optionally other reagents suitable for facilitating PCR reactions.
  • the invention also provides a reagent kit for detecting the presence of at least two pathogenic Candida species selected from the group consisting of C. glabrata, C. lusitaniae, C. guilliermondii, C. krusei, C. albicans, C. dubliniensis, C. tropicalis, C. parapsilosis and C. kefyr by PCR reaction, comprising at least 3 primers as defined in any of paragraphs designated a)-h) above and optionally other reagents suitable for facilitating PCR reactions.
  • the invention also provides a reagent kit for the genera specific detection of the presence of a Candida spe- cies, said species being advantageously selected from the group consisting of C. glabrata, C.
  • oligonucleotide primer pair having sequences selected from the group consisting of sequences identified by SEQ ID NOs 23 and 24, respectively, sequences being at least 90% identical with sequences identified by SEQ ID NOs 23 and 24, respectively, and complementary sequences thereof, and optionally other rea- gents suitable for facilitating PCR reactions, wherein SEQ ID NOs 23 and 24 are sequences surprisingly identified by the present inventors as being present in the ITS region of all the above defined and other tested Candida species but are missing from other related microorganisms tested so far.
  • the invention further concerns the use of an oligonucleotide having a sequence selected from the group consisting of sequences identified by any of SEQ ID NOs from 1 to 22, sequences being at least 90% identical with sequences identified by any of SEQ ID NOs from 1 to 22, and complementary sequences thereof for detecting the presence of at least one pathogenic Candida species selected from the group consisting of C. glabrata, C. lusitaniae, C. guilliermondii, C. krusei, C. albicans, C. dubliniensis, C. tropicalis, C. parapsilosis and C. kefyr in a biological sample.
  • the applied primer sequences are based on the Phospholi- pase-D gene region, accordingly, the invention further concerns the use of an oligonucleotide having a sequence selected from the group consisting of sequences identified by any of SEQ ID NOs from 11 to 22, sequences being at least 90% identical with sequences identified by any of SEQ ID NOs from 11 to 22 and complementary sequences thereof for detecting the presence of at least one pathogenic Candida species selected from the group consisting of C. glabrata, C. guilliermondii, C. krusei, C. albicans, C. dubliniensis, C. tropicalis, C. parapsilosis and C. kefyr in a biological sample.
  • the invention concerns the use of an oligonucleotide primer pair having sequences selected from the group consisting of sequences identified by SEQ ID NOs 23 and 24, respectively, sequences being at least 90% identical with sequences identified by SEQ ID NOs 23 and 24, respectively, and complementary sequences thereof for genera specific detection of a Candida species, said species being advan- tageously selected from the group consisting of C. glabrata, C. lusitaniae, C. guilliermondii, C. krusei, C. albicans, C. dubliniensis, C. tropicalis, C. parapsilosis and C. kefyr in a biological sample.
  • Figure 1 shows photographs of gel electrophoretic molecular weight determination of the Candida species specific amplicons produced using the species specific ITS based primer pair combinations of the invention.
  • Each photographed gel comprised the same markers and PCR reaction products produced on the same template DNAs in the same order in 16 lanes, as follows: lane 1: 1 kb marker (Promega); PCR reaction products comprising template DNA from the following species: lane 2 C. albicans; lane 3 C. lusitaniae; lane 4 C. krusei; lane 5 C. inconspicua; lane 6 C. dubliniensis; lane 7 C. tropicalis; lane 8 C. norvegensis; lane 9 C. pulcherima; lane 10 C.
  • Candida species are shown in the separate gel photographs: C. guillermondii (Fig. l.a); C. lusitaniae (Fig. l.b); C. glabrata (Fig. l.c); C. krusei (Fig. l.d); C. albicans (Fig. l.e); C. dubliniensis (Fig. l.f); C. tropicalis (Fig. l.g).
  • Figure 2 shows a photograph of gel electrophoretic molecular weight determination of the Candida species specific amplicons produced using the species specific primer pair combinations of the invention based on the Phospholipase-D gene region (see page 15).
  • the lanes of the gel photographed comprised the further marker and amplicons, respectively: ⁇ _ 100 bp marker; 2 C. krusei, 3_ C. glabrata, 4 C. dubliniensis, 5 C. guilliermondii, 6 C. tropicalis, 7 C. parapsilosis, 8 C. albicans, 9 C. kefyr.
  • FIG 3 shows photographs of gel electrophoretic experiments designed to demonstrate the applicability of the method of the invention for the species specific detection of Candida species when using cultivated clinical blood samples directly as PCR template material (without any prior purification or nucleic acid isolation step).
  • the results demonstrate that one Candida species specific PCR reaction mixture may comprise up to 3 ⁇ l heparin comprising blood sample which should comprise at least 100 CFU Candida cells, and the maximum heparin content in the reaction mixture may be around 0.05 U.
  • the applied Taq polymerase was non-heparin resistant, commercially available Taq DNA polymerase (Dupla-TaqTM, obtained from ZenonBio Ltd., Szeged, Hungary).
  • Example 1 Direct detection from cultivated blood samples: Two ml of blood sample from BD Vacutainer® Blood Collection Tubes containing heparin is cultivated for a two days period. The cultivation is carried out in heparin containing BD Vacutainer® tubes with 10 ml of YPD medium supplemented with the required antibiotics and 2 ml of the sample at 37 0 C on rotary shaker. After the incubation period 2 ⁇ l of the sample can be applied for direct detection. In this case there is no need for diluting the cultivated samples because in 3 ⁇ l sample the concentration of heparin is 0.05 IU.
  • the test can be carried out by using the WCsunF and WCsunR primer pairs, which were designed on the basis of the 18S rDNA region.
  • the detectable species are Candida albicans, C. dubliniensis, C. glabrata, C. guilliermodii, C. krusei, C. lusitaniae, C. parapsilosis and C. tropicalis.
  • the working combinations of the primers for identifying the species are: Canforl and Clusi for Candida lusitaniae, Canforl and Cguil for Candida guilliermondii, Canforl and Cglab for Candida glabrata, Canforl and Ckrus for Candida krusei, Canfor 2 and Cdubl for Candida dubliniensis, Canfor 2 and Ctropi for Candida tropicalis, Canfor 2 and Calbic for Candida albicans, Canfor 2 and Cpara for Candida parapsilosis.
  • Candida albicans C. dubliniensis, C. glabrata, C. guilliermodii, C. kefyr, C. lusitaniae, C. parapsilosis and C. tropicalis.
  • the working combinations of the primers for identifying the species are: CparkrD and CparaD for Candida parapsilosis, CparkrD and CkrusD for Candida krusei, CguitrD and CguiD for Candida guilliermondii, CguitrD and CtropD for Candida tropicalis, CglkefD and CglabD for Candida glabrata, CglkefD and CkefD for Candida kefyr, CalbduD and CaIbD for Candida albicans, CalbduD and CdubD for Candida dubliniensis.
  • PCR Introduce 1-3 ⁇ l of blood sample to a PCR tube containing 2 ⁇ l of buffer, 4 ⁇ l of dNTP mix, 3-3 ⁇ l of primers, 3,5 ⁇ l of MgCl 2, 0.2 ⁇ l of Taq polymerase (Dupla-TaqTM, obtained from ZenonBio Ltd., Szeged, Hungary), add bidistilled water to reach a final volume of 21,2 ⁇ l.
  • the PCR parameters for detecting the pathogens with primers based on the ITS region and with the whole- Candida primers are: preliminary denaturation step at 94°C for 30 seconds, 5 cycles of denaturation step at 94°C for 10 seconds, annealing/extension at 59°C for 20 seconds, 34 cycles of denaturation at 94°C for 10 seconds, annealing/extension at 61 0 C for 17 seconds.
  • the PCR parameters for detecting the pathogens with primers based on the Phospholipase-D are the following: preliminary denaturation step at 94°C for 30 seconds, 5 cycles of denaturation step at 94°C for 10 seconds, annealing/extension at 59°C for 20 seconds, 34 cycles of denaturation at 94°C for 10 seconds, annealing/extension at 64°C for 17 seconds.
  • Gel electrophoresis Prepare a 2% of agarose gel by adding 2 ml of 50-fold concentrated TAE buffer to 2 g of agarose and 98 ml of distilled water. Dissolve the agarose in the buffer by using a microwave oven. Run the gel on 60-80 mV for 20 minutes.
  • Example 2 Detection after cultivation with DNA extraction from blood samples: Two ml of blood sample from BD Vacutainer® Blood Collection Tubes containing heparin is cultivated for a two days period. The cultivation is carried out in heparin containing BD Vacutainer® tubes with 10 ml of YPD medium supplemented with the required antibiotics and 2 ml of the sample at 37 0 C on rotary shaker. After one day of incubation 1 ml of the sample is applied into a heparin free tube with 10 ml of YPD medium containing the required antibiotics and incubated at 37 0 C on rotary shaker for additional 24 hours.
  • the number of cells reaches the detectable amount which is 50-80 cells/PCR sample.
  • the cell content of the whole sample will be around 500000 CFU. Due to the dilutions the heparin content of the sample decreases to 1.4U/ml which is not able to inhibit the polymerase chain reaction.
  • DNA extraction protocol Apply 100 ⁇ l of the cultivated sample, 300 ⁇ l of 20 % N-lauroylsarcosine sodium salt, 300 ⁇ l of 5M ammonium acetate and 1 ⁇ l of ⁇ -mercaptoethanol into a microcentrifuge tube and vortex it 30 seconds. Incubate the mixture at 70 0 C for 15 minutes. After the incubation period vortex the sample again and add 600 ⁇ l of benzyl alcohol and 50 ⁇ l of benzyl benzoate. Centrifuge the mixture at room temperature at 13000 rpm for 15 minutes and pipet the supernatant into a new centrifuge tube.
  • the test can be carried out by using the WCsunF and WCsunR primer pairs, which were designed on the basis of the 18S rDNA region.
  • the detectable species are Candida albicans, C. dubliniensis, C. glabrata, C. guilliermodii, C. krusei, C. lusitaniae, C. parapsilosis and C. tropicalis.
  • the working combinations of the primers for identifying the species are: Canforl and Clusi for Candida lusitaniae, Canforl and Cguil for Candida guilliermondii, Canforl and Cglab for Candida glabrata, Canforl and Ckrus for Candida krusei, Canfor 2 and Cdubl for Candida dubliniensis, Canfor 2 and Ctropi for Candida tropicalis, Canfor 2 and Calbic for Candida albicans, Canfor 2 and Cpara for Candida parapsilosis. Determining eight Candida species using primer pairs based on the Phospholipase-D region. The detectable species are Candida albicans, C. dubliniensis, C. glabrata, C. guilliermodii, C.
  • the working combinations of the primers for identifying the species are: CparkrD and CparaD for Candida parapsilosis, CparkrD and CkrusD for Candida krusei, CguitrD and CguiD for Candida guilliermondii, CguitrD and CtropD for Candida tropicalis, CglkefD and CglabD for Candida glabrata, CglkefD and CkefD for Candida kefyr, CalbduD and CaIbD for Candida albicans, CalbduD and CdubD for Candida dubliniensis.
  • PCR Introduce 1-2 ⁇ l of sample to a PCR tube containing 2 ⁇ l of buffer, 4 ⁇ l of dNTP mix, 3-3 ⁇ l of primers, 2 ⁇ l of MgCl 2, 0.2 ⁇ l of Taq polymerase (Dupla-TaqTM, obtained from ZenonBio Ltd., Szeged, Hungary), add bidistilled water to reach a final volume of 21,2 ⁇ l.
  • Candida primers are: preliminary denaturation step at 94°C for 30 seconds, 5 cycles of denaturation step at 94°C for 10 seconds, annealing/extension at 57°C for 20 seconds, 34 cycles of denaturation at 94°C for 10 seconds, annealing/extension at 62°C for 17 seconds.
  • the PCR parameters for detecting the pathogens with primers based on the Phospholipase-D are the follow- ing: preliminary denaturation step at 94°C for 30 seconds, 5 cycles of denaturation step at 94°C for 10 seconds, annealing/extension at 59°C for 20 seconds, 34 cycles of denaturation at 94°C for 10 seconds, annealing/extension at 64°C for 17 seconds.
  • Gel electrophoresis Prepare a 2% of agarose gel by adding 2 ml of 50-fold concentrated TAE buffer to 2 g of agarose and 98 ml of distilled water. Dissolve the agarose in the buffer by using a microwave oven. Run the gel on 60-80 mV for 20 minutes.
  • Candida species from any commercially available solid state media which is suitable for culturing yeast (e.g. rice agar, CHROM-agar etc.). Suspend it in 100 ⁇ l distilled water. From this suspension 1 ⁇ l can be introduced to a PCR tube as the source of the template DNA.
  • yeast e.g. rice agar, CHROM-agar etc.
  • the detectable species are Candida albicans, C. dubliniensis, C. glabrata, C. guilliermodii, C. krusei, C. lusitaniae, C. parapsilosis and C. tropicalis.
  • the working combinations of the primers for identifying the species are: Canforl and Clusi for Candida lusitaniae, Canforl and Cguil for Candida guilliermondii, Canforl and Cglab for Candida glabrata, Canforl and Ckrus for Candida krusei, Canfor 2 and Cdubl for Candida dubliniensis, Canfor 2 and Ctropi for Candida tropicalis, Canfor 2 and Calbic for Candida albicans, Canfor 2 and Cpara for Candida parapsilosis.
  • Candida albicans C. dubliniensis, C. glabrata, C. guilliermodii, C. kefyr, C. lusitaniae, C. parapsilosis and C. tropicalis.
  • the working combinations of the primers for identifying the species are: CparkrD and CparaD for Candida parapsilosis, CparkrD and CkrusD for Candida krusei, CguitrD and CguiD for Candida guilliermondii, CguitrD and CtropD for Candida tropicalis, CglkefD and CglabD for Candida glabrata, CglkefD and CkefD for Candida kefyr, CalbduD and CaIbD for Candida albicans, CalbduD and CdubD for Candida dubliniensis.
  • PCR Introduce 1-2 ⁇ l of sample to a PCR tube containing 2 ⁇ l of buffer, 4 ⁇ l of dNTP mix, 3-3 ⁇ l of pri- mers, 2 ⁇ l of MgCl 2, 0.2 ⁇ l of Taq polymerase (Dupla-TaqTM, obtained from ZenonBio Ltd., Szeged, Hungary), add bidistilled water to reach a final volume of 21,2 ⁇ l.
  • the PCR parameters for detecting the pathogens with primers based on the ITS region and with the whole - Candida primers are: preliminary denaturation step at 94°C for 30 seconds, 5 cycles of denaturation step at 94°C for 10 seconds, annealing/extension at 57°C for 20 seconds, 34 cycles of denaturation at 94°C for 10 seconds, annealing/extension at 62°C for 17 seconds.
  • the PCR parameters for detecting the pathogens with primers based on the Phospholipase-D are the following: preliminary denaturation step at 94°C for 30 seconds, 5 cycles of denaturation step at 94°C for 10 seconds, annealing/extension at 59°C for 20 seconds, 34 cycles of denaturation at 94°C for 10 seconds, annealing/extension at 64°C for 17 seconds.
  • Gel electrophoresis Prepare a 2% of agarose gel by adding 2 ml of 50-fold concentrated TAE buffer to 2 g of agarose and 98 ml of distilled water. Dissolve the agarose in the buffer by using a microwave oven. Run the gel on 60-80 mV for 20 minutes.
  • Example 4 Direct identification from complex human samples excluding blood
  • the source of the samples could be body fluid, nails, skin, sputum, stool, swab of conjunctiva, throat, urine, or vaginal.
  • the samples are cultivated for a two days period. The cultivation is carried out in sterile Erlenmeyer flask or screw cap tube with 10 ml of YPD medium supplemented with the required antibiotics and the sample at 37 0 C on rotary shaker. After the incubation period 2 ⁇ l of the sample can be applied for direct detection. Detecting the presence of Candida in the sample: The test can be carried out by using the WCsunF and
  • WCsunR primer pairs which were designed on the basis of the 18S rDNA region.
  • the detectable species are Candida albicans, C. dubliniensis, C. glabrata, C. guilliermodii, C. krusei, C. lusitaniae, C. parapsilosis and C. tropicalis.
  • the working combinations of the primers for identifying the species are: Canforl and Clusi for Candida lusitaniae, Canforl and Cguil for Candida guilliermondii, Canforl and Cglab for Candida glabrata, Canforl and Ckrus for Candida krusei, Canfor 2 and Cdubl for Candida dubliniensis, Canfor 2 and Ctropi for Candida tropicalis, Canfor 2 and Calbic for Candida albicans, Canfor 2 and Cpara for Candida parapsilosis.
  • Candida albicans C. dubliniensis, C. glabrata, C. guilliermodii, C. kefyr, C. lusitaniae, C. parapsi- losis and C. tropicalis.
  • the working combinations of the primers for identifying the species are: CparkrD and CparaD for Candida parapsilosis, CparkrD and CkrusD for Candida krusei, CguitrD and CguiD for Candida guilliermondii, CguitrD and CtropD for Candida tropicalis, CglkefD and CglabD for Candida glabrata, CglkefD and CkefD for Candida kefyr, CalbduD and CaIbD for Candida albicans, CalbduD and CdubD for Candida dubliniensis.
  • PCR Introduce 1-2 ⁇ l of sample to a PCR tube containing 2 ⁇ l of buffer, 4 ⁇ l of dNTP mix, 3-3 ⁇ l of primers, 2.5 ⁇ l of MgCU , 0.2 ⁇ l of Taq polymerase (Dupla-TaqTM, obtained from ZenonBio Ltd., Szeged, Hungary), add bidistilled water to reach a final volume of 21,2 ⁇ l.
  • the PCR parameters for detecting the pathogens with primers based on the ITS region and with the whole - Candida primers are: preliminary denaturation step at 94°C for 30 seconds, 5 cycles of denaturation step at 94°C for 10 seconds, annealing/extension at 57°C for 20 seconds, 34 cycles of denaturation at 94°C for 10 seconds, annealing/extension at 62°C for 17 seconds.
  • the PCR parameters for detecting the pathogens with primers based on the Phospholipase-D are the following: preliminary denaturation step at 94°C for 30 seconds, 5 cycles of denaturation step at 94°C for 10 seconds, annealing/extension at 59°C for 20 seconds, 34 cycles of denaturation at 94°C for 10 seconds, anneal- ing/extension at 64°C for 17 seconds.
  • Gel electrophoresis Prepare a 2% of agarose gel by adding 2 ml of 50-fold concentrated TAE buffer to 2 g of agarose and 98 ml of distilled water. Dissolve the agarose in the buffer by using a microwave oven. Run the gel on 60-80 mV for 20 minutes.
  • Example 5 Detection from complex human samples excluding blood with DNA extraction from the samples
  • the source of the samples can be body fluid, nails, skin, sputum, stool, swab of conjunctiva, throat, urine, or vaginal.
  • the samples are cultivated for a two days period. The cultivation is carried out in sterile Erlenmeyer flask or screw cap tube with 10 ml of YPD medium supplemented with the required antibiotics and the sample at 37 0 C on rotary shaker.
  • DNA extraction protocol Apply 100 ⁇ l of the cultivated sample, 300 ⁇ l of 20 % N-lauroylsarcosine sodium salt, 300 ⁇ l of 5M ammonium acetate and 1 ⁇ l of ⁇ -mercaptoethanol into a microcentrifuge tube and vortex it 30 seconds.
  • the test can be carried out by using the WCsunF and WCsunR primer pairs, which were designed on the basis of the 18S rDNA region.
  • the detectable species are Candida albicans, C. dubliniensis, C. glabrata, C. guilliermodii, C. krusei, C. lusitaniae, C. parapsilosis and C. tropicalis.
  • the working combinations of the primers for identifying the species are: Canforl and Clusi for Candida lusitaniae, Canforl and Cguil for Candida guilliermondii, Canforl and Cglab for Candida glabrata, Canforl and Ckrus for Candida krusei, Canfor 2 and Cdubl for Candida dubliniensis, Canfor 2 and Ctropi for Candida tropicalis, Canfor 2 and Calbic for Candida albicans, Canfor 2 and Cpara for Candida parapsilosis.
  • Candida albicans C. dubliniensis, C. glabrata, C. guilliermondii, C. kefyr, C. lusitaniae, C. parapsilosis and C. tropicalis.
  • the working combinations of the primers for identifying the species are: CparkrD and CparaD for Candida parapsilosis, CparkrD and CkrusD for Candida krusei, CguitrD and CguiD for Candida guilliermondii, CguitrD and CtropD for Candida tropicalis, CglkefD and CglabD for Candida glabrata, CglkefD and CkefD for Candida kefyr, CalbduD and CaIbD for Candida albicans, CalbduD and CdubD for Candida dubliniensis.
  • PCR Introduce 1-2 ⁇ l of sample to a PCR tube containing 2 ⁇ l of buffer, 4 ⁇ l of dNTP mix, 3-3 ⁇ l of pri- mers, 2 ⁇ l of MgCl 2, 0.2 ⁇ l of Taq polymerase (Dupla-TaqTM, obtained from ZenonBio Ltd., Szeged, Hungary), add bidistilled water to reach a final volume of 21,2 ⁇ l.
  • the PCR parameters for detecting the pathogens with primers based on the ITS region and with the whole - Candida primers are: preliminary denaturation step at 94°C for 30 seconds, 5 cycles of denaturation step at 94°C for 10 seconds, annealing/extension at 57°C for 20 seconds, 34 cycles of denaturation at 94°C for 10 seconds, annealing/extension at 62°C for 17 seconds.
  • the PCR parameters for detecting the pathogens with primers based on the Phospholipase-D are the following: preliminary denaturation step at 94°C for 30 seconds, 5 cycles of denaturation step at 94°C for 10 seconds, annealing/extension at 59°C for 20 seconds, 34 cycles of denaturation at 94°C for 10 seconds, annealing/extension at 64°C for 17 seconds.
  • Gel electrophoresis Prepare a 2% of agarose gel by adding 2 ml of 50-fold concentrated TAE buffer to 2 g of agarose and 98 ml of distilled water. Dissolve the agarose in the buffer by using a microwave oven. Run the gel on 60-80 mV for 20 minutes.
  • Example 6 Multiplex Polymerase Chain Reaction
  • the multiplex PCR can be performed directly from the cultivated samples, or by using the DNA extract.
  • the primer Canforl can be combined in one reaction with C. glabrata (Cglab) and C. lusitaniae (Clusi) specific primers, or with the C. guilliermondii
  • the primer Canfor2 can be combined in the same PCR tube with
  • C. albicans Calbic
  • C. dubliniensis Cdubl
  • Ctropi C. tropicalis
  • Cpara C. pa- rapsilosis
  • the working combinations of the primers designed on the basis of the Phospholipase-D region for the multiplex PCR are: CparkrD with the Candida parapsilosis (CparaD) and Candida krusei (CkrusD) specific primers. CguitrD with the Candida guilliermondii (CguiD) and Candida tropicalis (CtropD) specific primers. CglkefD with the Candida glabrata (CglabD) and Candida kefyr (CkefD) specific primers. CalbduD with the Candida albicans (CaIbD) and Candida dubliniensis (Cdub) specific primers.
  • PCR for ITS based primers Introduce 2 ⁇ l of sample to a PCR tube containing 2 ⁇ l of buffer, 4 ⁇ l of dNTP mix, 3 ⁇ l of the shared (Canforl or Canfor2) primers, 2-2 ⁇ l of the species specific primers, 2.4 ⁇ l of MgCl 2, 0.2 ⁇ l of Taq polymerase (Dupla-TaqTM, obtained from ZenonBio Ltd., Szeged, Hungary), add bidistilled water to reach a final volume of 21,2 ⁇ l.
  • Candida primers are: preliminary denaturation step at 94°C for 30 seconds, 5 cycles of denaturation step at 94°C for 10 seconds, annealing/extension at 57°C for 20 seconds, 34 cycles of denaturation at 94°C for 10 seconds, annealing/extension at 60 0 C for 17 seconds.
  • PCR for Phospholipase-D based primers Introduce 1-2 ⁇ l of sample to a PCR tube containing 2 ⁇ l of buffer, 4 ⁇ l of dNTP mix, 4 ⁇ l of the shared (CparkrD, CguitrD, CglkefD, CalbduD) primers, 2-2 ⁇ l of the species specific primers, 2.6 ⁇ l Of MgCl 2, 2 0.2 ⁇ l of Taq polymerase (Dupla-TaqTM, obtained from ZenonBio Ltd., Szeged, Hungary), add bidistilled water to reach a final volume of 21,2 ⁇ l.
  • the PCR parameters for detecting the pathogens with primers based on the Phospholipase-D are the following: preliminary denaturation step at 94°C for 30 seconds, 5 cycles of denaturation step at 94°C for 10 seconds, annealing/extension at 58°C for 20 seconds, 34 cycles of denaturation at 94°C for 10 seconds, annealing/extension at 64°C for 17 seconds.
  • Neomycin stock solution (10mg/ml)
  • Chloramphenicol stock solution (34mg/ml)
  • the final concentration of the antibiotics in the case of Neomycin must be in the range of 10-80 ⁇ l/ml and for Chloramphenicol in the range of 0,7-5 ⁇ l/ml.
  • the mixture is vortex-mixed for 30 second, then incubated at 70 0 C for 15 minutes.
  • the sample is vortex-mixed again
  • the following step is the addition of 600 ⁇ l of benzyl alcohol and 50 ⁇ l of benzyl benzoate.
  • the mixture is centrifuged at room temperature at 13000 rpm for 15 minutes and the supernatant is pipetted into a new centrifuge tube.
  • the supernatant is precipitated with 650 ⁇ l of isopropyl alcohol and centrifuged again at 13000 rpm for 10 minutes.
  • the supernatant is discarded and the pellet is washed with 650 ⁇ l of 70 % ethanol, then centrifuged again at 13000 rpm for 5 minutes. Ethanol is discarded and the sample is dried in a vacuum dryer.
  • the DNA is resuspended in 30 ⁇ l of bidistilled water and stored at -20 0 C.
  • dNTP mix 960 ⁇ l of bidistilled water and 10-10 ⁇ l of 100 mM dATP, dCTP, dGTP and dTTP
  • the final volume of the reaction is 21.2 ⁇ l 2 ⁇ l of puffer 4 ⁇ l of dNTP mix 4-2-2 ⁇ l of primers 1,6-3.5 ⁇ l OfMgCl 2
  • Protocol for direct PCR from blood samples The final volume of the reaction is 21.2 ⁇ l 2 ⁇ l of puffer 4 ⁇ l ofdNTP mix 3-3 ⁇ l of primers 3.4-4.7 ⁇ l OfMgCl 2 0.2 ⁇ l of Taq
  • Ethidium bromide 5 ⁇ l for 100ml of TAE buffer from a 10 mg/ml stock solution
  • the electrophoresis can be carried out on 60- 80 mV for 20 minutes.
  • C. dubliniensis fragment length 344 bp
  • C. tropicalis fragment length 398 bp
  • C. albicans fragment length 454 bp
  • Fragment length -480 bp
  • CkefD forward 5 ' - CTGGTCATTG AGCG AAGCTCT - 3 ' SEQ ID NO: 19
  • CalbduD shared reverse 5 ' - CCTTCTCT ATGTGCTCG AATG A - 3 ' SEQ ID NO: 20
  • CdubD forward 5 ' - C ATGGACTAG ATGGT ACCTGC - 3 ' SEQ ID NO: 21
  • C. albicans fragment length: 475 bp
  • C. dubliniensis fragment length: 242 bp
  • Candida dubliniensis Fungemia the First Four Cases in North America. Emerging Infectious Diseases, 6, 1. Fidel Jr. P. L., Vazquez J. A., Sobel J. D. (1999).
  • Candida glabrata Review of Epidemiology, Pathogenesis, and Clinical Disease with Comparison to C. albicans. Clinical Microbiology Reviews, 12, 80-96.
  • Candida parapsilosis Emergence of 310 Candida parapsilosis as the predominant species causing candidemia in children. Clin Infect Dis 26, 1086-1088. Meis J. F. G. M., Ruhnke M., De Pauw B. E., Odds F. C, Siegert W. and Verweij P. E. (1999). Candida dubliniensis Candidemia in Patients with Chemotherapy -Induced Neutropenia and Bone Marrow Transplantation Emerging Infectious Diseases, 5, 1.

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Abstract

The invention is concerned with the field of PCR based diagnostic methods, more specifically with the di- agnosis of infectious diseases caused by different Candida species. Still more specifically the invention provides PCR based diagnostic methods for the detection of Candida species most commonly causing invasive candidia- sis. The methods of the invention enable the detection of the Candida species of interest directly from heparin containing blood samples and the specific PCR primers provided by the invention also enable the detection of more than one pathogenic Candida species in one single PCR reaction based on the significantly different sizes of the species specific amplicons produced.

Description

PCR based diagnostic method for the identification of clinically important Candida species
FIELD OF THE INVENTION
The present invention is concerned with the field of PCR based diagnostic methods, more specifically with the diagnosis of infectious diseases caused by different Candida species. Still more specifically the invention provides PCR based diagnostic methods for the detection of Candida species most commonly causing invasive candidiasis. The methods of the invention advantageously enable the detection of the Candida species of interest directly from heparin containing blood samples and the specific PCR primers provided by the invention also enable the detection of more than one pathogenic Candida species in one single PCR reaction based on the significantly different sizes of the species specific amplicons produced.
BACKGROUND OF THE INVENTION
Infections caused by Candida species are widespread throughout the world. Although Candida albicans is the most common Candida species encountered as the causal agent of human infections, other Candida species have also been increasingly associated with disseminated disease since the 1990s.
The genus Candida includes around 154 species. Among these, six are most frequently isolated in the case of human infections. While Candida albicans is the most abundant and significant species, Candida tropicalis, Candida glabrata, Candida parapsilosis, Candida krusei, and Candida lusitaniae are also isolated as causative agents of Candida infections. Importantly, there has been a recent increase in infections due to non -albicans Candida spp., such as Candida glabrata and Candida krusei.
On the basis of the data obtainable from the commonly used "Dr. Fungus" homepage (www.doctorfungus.org), the following species of Candida are most commonly causing invasive candidiasis: Candida albicans (frequency: 50%) Candida tropicalis (15-30%) Candida glabrata (15-30%) Candida parapsilosis (15-30%) Candida krusei (-1%) Candida lusitaniae (-1%) Among them, C. parapsilosis is the second most common yeast species isolated from blood stream infections as reported in several surveys (Safdar et al., 2002; Messer et al., 2006; Bassetti et al., 2006). This species has emerged as an important nosocomial pathogen, with clinical manifestations including fungemia, endocarditis, endophthalmitis, septic arthritis, and peritonitis, and usually occur in association with invasive procedures or prosthetic devices (Weems, 1992). In some children's hospitals, C. parapsilosis became the predominant species causing candidemia (Levy et al., 1998). This species is more frequent in bloodstream infections of neonates, in transplant recipients, and in patients who received parenteral nutrition or previous antifungal therapy (Almirante et al., 2006). This species is also frequently associated with catheter-associated candidemia and intravenous hyperalimentation (Kremery & Barnes, 2002).
Candida krusei is inherently resistant to fluconazole and is emerging as a frequent causal agent of fungemia in patients with hematologic malignant neoplasms. In neutropenic patients, (Abbas et al. 2000) C. krusei fungemia results in high mortality.
To date, Candida glabrata was considered as a relatively non-pathogenic commensal fungal organism of human mucosal tissues (Fidel et al.). However, with the increased use of immunosuppressive agents, mucosal and systemic infections caused by C. glabrata have increased significantly, especially in the human immunodefi- ciency virus-infected population. A major obstacle in C. glabrata infections is their innate resistance to azole an- timycotic therapy, which is very effective in treating infections caused by other Candida species. Candida gla- brata, formerly known as Torulopsis glabrata, contrasts with other Candida species in its nondimorphic blasto- conidial morphology and haploid genome. C. glabrata is currently ranked second or third as the causative agent of superficial (oral, esophageal, vaginal, or urinary) or systemic Candida infections, which are often nosocomial. Candida tropicalis is a major cause of septicemia and disseminated candidiasis, especially in patients with lymphoma, leukemia and diabetes. It is the second most frequently encountered medical pathogen, next to C. albicans, and is also found as part of the normal human mucocutaneous flora. Sucrose negative variants of C. tropicalis have also been increasingly found in cases of disseminated candidiasis. Environmental isolations have been made from faeces, shrimp, kefir, and soil. Candida lusitaniae is known for amphotericin B resistance (Peyron et al. 2000). The first report of fatal infection occurred in a neutropenic patient where the initial isolate was susceptible to amphotericin B but subsequent isolates had developed amphotericin B resistance. It is well-documented that this species is able to develop resistance, but some strains display resistance prior to amphotericin B therapy. Although amphotericin B resistance is a concern, reports exist where amphotericin B has been used to successfully treat fungemia in severely immunocompromised patients.
Although a rare cause of invasive candidiasis, Candida guilliermondii has been reported to exhibit decreased susceptibility to antifungal agents (Girmenia et al. 2006). Aside from case reports and small surveys, there is little information regarding the epidemiology and antifungal susceptibility profile of C. guilliermondii. Candida guilliermondii is infrequently isolated from blood cultures (1 to 5%), and infections caused by this spe- cies have been reported in cancer, surgical, and intensive care unit patients, including a pseudo-outbreak of can- didemia in a neonatal intensive care unit. Malignancy, neutropenia, and bone marrow transplantation have been reported as risk factors for acquiring C. guilliermondii infections. Treatment of these infections may present problems, especially for immunocompromised patients; a high percentage of strains have diminished susceptibility to fluconazole. Candida kefyr is a rare cause of candidiasis and is usually associated with superficial cutaneous manifestations rather than systemic disease. It has been isolated from nails and lung infections. Environmental isolations have been made from cheese and dairy products. C. kefyr infections reflect the increase in Candida species as responsible of disseminated infections in immunodepressed patients.
In recent years, Candida species other than C. albicans have emerged as causes of human candidiasis, par- ticularly in HIV-infected and other immunocompromised persons. C. dubliniensis, a recently described species closely related to C. albicans, has been implicated as an agent of oral candidiasis in HIV-positive persons but has also been recovered from HIV-negative persons with clinical signs of oral candidiasis and from the genital tract of some women with vaginitis. First isolated from AIDS patients in Dublin, Ireland, C. dubliniensis has a worldwide distribution. Most isolates are susceptible to amphotericin B and the azoles, but resistance has been shown in HIV-positive patients on fluconazole for oral candidiasis (Brandt et al. 2000, Meis et al. 1999). Its potential to cause deep or disseminated candidiasis is not known, largely because C. dubliniensis has rarely been isolated from sterile body sites; however, the phenotypic characteristics the organism shares with C. albicans (producing germ tubes and chlamydospores) suggest that some C. dubliniensis isolates may have been misidenti- fied as C. albicans. The above mentioned features of these Candida species clearly show the importance of a fast and reliable species identification method.
There are numerous techniques in the art having been developed to discriminate among human pathogenic Candida species. However, in spite of the continuous efforts in this field all presently used methodologies have serious drawbacks.
Microscopic investigation of the clinical isolates is not species specific; this is only a supplementary tool even using the best staining techniques.
ELISA techniques available for routine diagnostics are not selective enough, and are restricted mainly to C. albicans. There are several approaches for PCR -based detection of Candida species, however, most of these do not satisfy the specific requirements of a clinical laboratory and do not cover all the clinically important species.
One of the most wide-spread identification method used by clinical laboratories is the application of selective media, particularly CHROM-agar(s). However, these techniques are time consuming, furthermore, the morphological characteristics used as a base of identification, such as the color of the colonies, the structure of their surface, the presence or absence of pseudohyphae is not unequivocal, and therefore can easily lead to misidenti- fication. Another problem with such approaches is that the growing colony might represent more than one species. According to the above described problems the mucosal swabs give unequivocal results only in 50-60 per- cents of the cases, while in the case of epithelial scrapings, excrements, blood samples the rate is much lower, practically only the positive result can be considered as valid. It could be seen that the currently used methods do not cope with the needs of the clinical species identification and often lead to false results.
Furthermore, it is frequently experienced in the case of invasive candidiasis that the infection is caused by more than one Candida species one or more of which might be highly overwhelming. Considering the above - detailed information regarding the fact that different Candida species may react very differently to any applied specific anti-fungi therapy, a person skilled in the pertinent art will easily comprehend that it is clinically very important to reliably identify all the different Candida species being present in an individual clinical sample. This is because if the applied therapy is only effective against the predominant (and therefore most easily identifiable) causative agent, other secondary pathogenic species present may result in the outlasting of the disease even if the predominant causative agent is successfully eliminated. It should therefore be clear from all what was said above, that there is a significant need in the art for a rapid, sensitive and complex diagnostic method that is capable of the reliable detection and identification of all the different Candida species that may be expectably present in a Candida infection even if said species being present in a significantly minor amount as compared to other species being also present.
The object of the present invention is therefore to provide a reliable, rapid, cost effective and complex diag- nostic method and reagent kit suitable for the identification of all the different Candida species so far identified as significant causative agents of invasive candidiasis.
Furthermore, the person skilled in the pertinent art will also easily comprehend, considering that blood is one of the clinically most important samples for detecting pathogenic Candida species, that when developing a user friendly, clinically applicable PCR based method for the detection of pathogenic Candida species, it can be a major advantage if the PCR reaction can be performed directly using clinical blood samples as PCR templates - A - without performing any purification steps. Therefore, a further object of this invention is to provide a simple user friendly clinically applicable PCR based method and reagent kit for the detection of all clinically relevant pathogenic Candida species directly using clinical blood samples as PCR templates.
SUMMARY OF THE INVENTION
The present invention provides a rapid and cost effective method for identifying nine different Candida species from complex clinical samples, using a unique species specific PCR reaction. The samples can be derived from any solid state media, epithelial or nail scrapings, excrement, mucosal samples or cultivated blood samples.
The versatility of this method has been increased by the possibility to carry out this identification by intro- ducing the clinical blood sample directly into the PCR tube, or making a unique DNA extraction step if there is a need for further investigation or the samples have to be stored economically.
Even though the pertinent art definitely teaches that commercially available unmodified Taq polymerases are not suitable for using them in PCR reactions when heparinized clinical blood samples are directly used as target material because of their sensitivity to heparin [Al-Soud W. A. and Radstrom P. (2001); Yokota, M., et al. (1999)], we have surprisingly found that in specifically defined circumstances, heparinized clinical blood samples may be directly used as PCR template in highly sensitive Candida specific PCR reactions even when using unmodified, non-heparin resistant Taq polymerase.
DETAILED DESCRIPTION OF THE INVENTION The present invention, therefore, provides a PCR based method for detecting the presence of at least one pathogenic Candida species present in a heparin containing blood sample, wherein a part of said heparin containing blood sample, comprising material serving as PCR template, is directly added to a Candida species specific PCR reaction mixture. The method of the invention is advantageously performed using a non-heparin resistant Taq DNA polymerase. The expression "non-heparin resistant Taq DNA polymerase" in the context of the present in- vention is used to define any commercially available Taq DNA polymerase that is not commercialized or described in the art as being specially heparin resistant.
The blood sample used in the method of the invention, advantageously, prior to the addition of a part thereof to the PCR reaction mixture, is preincubated, advantageously for 1 -3 days, in conditions facilitating the growth of said at least one pathogenic Candida species being supposedly present in said blood sample, in order to en- hance the sensitivity of the method of the invention. Said preincubation is advantageously done in the presence of at least one antibiotic, advantageously Neomycin and/or Chloramphenicol.
The PCR reaction mixture in accordance with the present invention advantageously contains less than 0.06 IU heparin, more advantageously less than 0.05 IU and a magnesium ion concentration of at least 4 mM, advantageously at least 5 mM. The method of the invention is advantageously useful for the detection of pathogenic Candida species selected from the group consisting of C. glabrata, C. lusitaniae, C. guilliermondii, C. krusei, C. albicans, C. dubli- niensis, C. tropicalis, C. parapsilosis and C. kefyr. The specific primer pairs of the present invention are designed in a way that the pathogenic Candida species to be identified are possible to identify on the basis of the size of the amplicon produced in the species specific PCR reaction mixture. The specifically designed primer pairs used in the present invention are designed to be capable of specifically hybridizing either to a sequence re- gion present in the internal transcribed spacer (ITS) region of the ribosomal gene repeat or to a species specific sequence region present in the Phospholipase-D gene of said at least one pathogenic Candida species, which gene was surprisingly identified by the present inventors as being useful for the species specific identification of all so far examined pathogenic Candida species of interest. More specifically, in the method of the invention the Candida species specific PCR primer pairs applied are selected from the group consisting of sequences identified by any of SEQ ID NOs from 1 to 22, sequences at least 90% identical with sequences identified by any of SEQ ID NOs from 1 to 22, and complementary sequences thereof.
In an advantageous embodiment of the present invention the Candida species specific PCR reaction mixture comprises more than one, advantageously more than two PCR primer pairs being specific for more than one, advantageously more than two Candida species, and wherein said primers, upon suitable thermal cycling, enable the production of more than one, advantageously more than two species specific amplicons each having a characteristic individual size being gel-electrophoretically distinguishable from any other Candida species specific amplicon that may be produced in said Candida species specific PCR reaction mixture. In a further advantageous embodiment of the present invention some of the individual PCR primers being present in said Candida species specific PCR reaction mixture are designed to be capable of specifically hybridizing to target sequences originating from more than one, advantageously more than two different Candida species being supposedly present in said blood or other biological sample, reducing thereby the overall number of different specific primers needed in the Candida species specific PCR reactions of the invention, where more than one pathogenic Candida species is identified within one single specific PCR reaction.
In a further advantageous embodiment of the present invention, the presence of two pathogenic Candida species selected from the group consisting of C. glabrata, C. lusitaniae, C. guilliermondii, C. krusei, C. albicans, C. dubliniensis, C. tropicalis, C. parapsilosis and C. kefyr is detected in one single Candida species specific PCR reaction, wherein said primers applied in said one single Candida species specific PCR reaction are having the sequences identified by a) SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 4 for C. glabrata and C. lusitaniae; or b) SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5 for C. guilliermondii and C. krusei; or c) SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 9 for C. albicans and C. dubliniensis; or d) SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10 for C. tropicalis and C. parapsilosis; or e) SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13 for C. parapsilosis and C. krusei; or f) SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 for C. guilliermondii and C. tropicalis; or g) SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19 for C. glabrata and C. kefyr; or h) SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22 for C. dubliniensis and C. albicans; or sequences being at least 90% identical with sequences defined in the above paragraphs a)-h) or complemen- tary sequences thereof, and wherein the identification of said two pathogenic Candida species is advantageously done based on the sizes of their species specific amplicons produced in said single species specific PCR reaction, advantageously determined by gel electrophoresis.
The present inventors have surprisingly found for the first time that specific sequences present in the Phospholipase-D gene of different pathogenic Candida species of interest enable the designing of species specific primer pairs hybridizing to said species specific sequence regions and capable of giving rise thereby to species specific PCR amplicons the size of which being characteristic to said species of interest.
The present invention, therefore, further provides a PCR based method for detecting the presence of at least one pathogenic Candida species present in a biological sample, wherein the Candida species specific PCR pri- mer pair(s) applied is(are) designed to be capable of specifically hybridizing to a species specific sequence region present in the Phospholipase-D gene of said at least one pathogenic Candida species. The sequences of said Phospholipase-D specific primers of the invention are advantageously selected from a group consisting of sequences identified by any of SEQ ID NOs from 11 to 22, sequences at least 90% identical with sequences identified by any of SEQ ID NOs from 11 to 22, and complementary sequences thereof. The expression "PCR based method for detecting the presence" of a microorganism is used herein to identify any method that comprises at least one step wherein a specific nucleic acid sequence present in a target microorganism is amplified by at least one PCR reaction, and then said amplified specific sequence is identified.
The invention further provides reagent kits for detecting the presence of at least one pathogenic Candida species selected from the group consisting of C. glabrata, C. lusitaniae, C. guilliermondii, C. krusei, C. albicans, C. dubliniensis, C. tropicalis, C. parapsilosis and C. kefyr by PCR reaction, comprising at least one primer pair wherein each primer having a sequence selected from the group consisting of sequences identified by any of SEQ ID NOs from 1 to 22, sequences being at least 90% identical with sequences identified by any of SEQ ID NOs from 1 to 22, and complementary sequences thereof and optionally other reagents suitable for facilitating PCR reactions. The invention also provides a reagent kit for detecting the presence of at least two pathogenic Candida species selected from the group consisting of C. glabrata, C. lusitaniae, C. guilliermondii, C. krusei, C. albicans, C. dubliniensis, C. tropicalis, C. parapsilosis and C. kefyr by PCR reaction, comprising at least 3 primers as defined in any of paragraphs designated a)-h) above and optionally other reagents suitable for facilitating PCR reactions. The invention also provides a reagent kit for the genera specific detection of the presence of a Candida spe- cies, said species being advantageously selected from the group consisting of C. glabrata, C. lusitaniae, C. guilliermondii, C. krusei, C. albicans, C. dubliniensis, C. tropicalis, C. parapsilosis and C. kefyr by PCR reaction, comprising an oligonucleotide primer pair having sequences selected from the group consisting of sequences identified by SEQ ID NOs 23 and 24, respectively, sequences being at least 90% identical with sequences identified by SEQ ID NOs 23 and 24, respectively, and complementary sequences thereof, and optionally other rea- gents suitable for facilitating PCR reactions, wherein SEQ ID NOs 23 and 24 are sequences surprisingly identified by the present inventors as being present in the ITS region of all the above defined and other tested Candida species but are missing from other related microorganisms tested so far.
The invention further concerns the use of an oligonucleotide having a sequence selected from the group consisting of sequences identified by any of SEQ ID NOs from 1 to 22, sequences being at least 90% identical with sequences identified by any of SEQ ID NOs from 1 to 22, and complementary sequences thereof for detecting the presence of at least one pathogenic Candida species selected from the group consisting of C. glabrata, C. lusitaniae, C. guilliermondii, C. krusei, C. albicans, C. dubliniensis, C. tropicalis, C. parapsilosis and C. kefyr in a biological sample.
In an advantageous embodiment of the invention, the applied primer sequences are based on the Phospholi- pase-D gene region, accordingly, the invention further concerns the use of an oligonucleotide having a sequence selected from the group consisting of sequences identified by any of SEQ ID NOs from 11 to 22, sequences being at least 90% identical with sequences identified by any of SEQ ID NOs from 11 to 22 and complementary sequences thereof for detecting the presence of at least one pathogenic Candida species selected from the group consisting of C. glabrata, C. guilliermondii, C. krusei, C. albicans, C. dubliniensis, C. tropicalis, C. parapsilosis and C. kefyr in a biological sample.
In a further advantageous embodiment, the invention concerns the use of an oligonucleotide primer pair having sequences selected from the group consisting of sequences identified by SEQ ID NOs 23 and 24, respectively, sequences being at least 90% identical with sequences identified by SEQ ID NOs 23 and 24, respectively, and complementary sequences thereof for genera specific detection of a Candida species, said species being advan- tageously selected from the group consisting of C. glabrata, C. lusitaniae, C. guilliermondii, C. krusei, C. albicans, C. dubliniensis, C. tropicalis, C. parapsilosis and C. kefyr in a biological sample.
BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 shows photographs of gel electrophoretic molecular weight determination of the Candida species specific amplicons produced using the species specific ITS based primer pair combinations of the invention. Each photographed gel comprised the same markers and PCR reaction products produced on the same template DNAs in the same order in 16 lanes, as follows: lane 1: 1 kb marker (Promega); PCR reaction products comprising template DNA from the following species: lane 2 C. albicans; lane 3 C. lusitaniae; lane 4 C. krusei; lane 5 C. inconspicua; lane 6 C. dubliniensis; lane 7 C. tropicalis; lane 8 C. norvegensis; lane 9 C. pulcherima; lane 10 C. glabrata; lane 11 C. parapsilosis; lane 12 C. guilliermodi; lane 13 C. zeylanoides; lane 14 C. lipolitica; lane 15 C. norvegica; lane 16 100 bp marker or 1 kb marker (Promega). Specific detection of the following Candida species are shown in the separate gel photographs: C. guillermondii (Fig. l.a); C. lusitaniae (Fig. l.b); C. glabrata (Fig. l.c); C. krusei (Fig. l.d); C. albicans (Fig. l.e); C. dubliniensis (Fig. l.f); C. tropicalis (Fig. l.g).
Figure 2 shows a photograph of gel electrophoretic molecular weight determination of the Candida species specific amplicons produced using the species specific primer pair combinations of the invention based on the Phospholipase-D gene region (see page 15). The lanes of the gel photographed comprised the further marker and amplicons, respectively: \_ 100 bp marker; 2 C. krusei, 3_ C. glabrata, 4 C. dubliniensis, 5 C. guilliermondii, 6 C. tropicalis, 7 C. parapsilosis, 8 C. albicans, 9 C. kefyr.
Figure 3 shows photographs of gel electrophoretic experiments designed to demonstrate the applicability of the method of the invention for the species specific detection of Candida species when using cultivated clinical blood samples directly as PCR template material (without any prior purification or nucleic acid isolation step). The results demonstrate that one Candida species specific PCR reaction mixture may comprise up to 3μl heparin comprising blood sample which should comprise at least 100 CFU Candida cells, and the maximum heparin content in the reaction mixture may be around 0.05 U. The applied Taq polymerase was non-heparin resistant, commercially available Taq DNA polymerase (Dupla-Taq™, obtained from ZenonBio Ltd., Szeged, Hungary).
EXAMPLES
Example 1: Direct detection from cultivated blood samples: Two ml of blood sample from BD Vacutainer® Blood Collection Tubes containing heparin is cultivated for a two days period. The cultivation is carried out in heparin containing BD Vacutainer® tubes with 10 ml of YPD medium supplemented with the required antibiotics and 2 ml of the sample at 37 0C on rotary shaker. After the incubation period 2 μl of the sample can be applied for direct detection. In this case there is no need for diluting the cultivated samples because in 3 μl sample the concentration of heparin is 0.05 IU. This concentration surprisingly does not noticeably affect the reaction, if the concentration of magnesium is increased to at least 4 mM in the reaction mixture, even when using commercially available non-heparin resistant Taq DNA polymerase (i.e. Dupla-Taq™, obtained from ZenonBio Ltd., Szeged, Hungary).
Detecting the presence of Candida in the sample: The test can be carried out by using the WCsunF and WCsunR primer pairs, which were designed on the basis of the 18S rDNA region.
Determination of eight Candida species using primer pairs based on the ITS region: The detectable species are Candida albicans, C. dubliniensis, C. glabrata, C. guilliermodii, C. krusei, C. lusitaniae, C. parapsilosis and C. tropicalis. The working combinations of the primers for identifying the species are: Canforl and Clusi for Candida lusitaniae, Canforl and Cguil for Candida guilliermondii, Canforl and Cglab for Candida glabrata, Canforl and Ckrus for Candida krusei, Canfor 2 and Cdubl for Candida dubliniensis, Canfor 2 and Ctropi for Candida tropicalis, Canfor 2 and Calbic for Candida albicans, Canfor 2 and Cpara for Candida parapsilosis.
Determining eight Candida species using primer pairs based on the Phospholipase-D region. The detectable species are Candida albicans, C. dubliniensis, C. glabrata, C. guilliermodii, C. kefyr, C. lusitaniae, C. parapsilosis and C. tropicalis. The working combinations of the primers for identifying the species are: CparkrD and CparaD for Candida parapsilosis, CparkrD and CkrusD for Candida krusei, CguitrD and CguiD for Candida guilliermondii, CguitrD and CtropD for Candida tropicalis, CglkefD and CglabD for Candida glabrata, CglkefD and CkefD for Candida kefyr, CalbduD and CaIbD for Candida albicans, CalbduD and CdubD for Candida dubliniensis.
PCR: Introduce 1-3 μl of blood sample to a PCR tube containing 2 μl of buffer, 4 μl of dNTP mix, 3-3 μl of primers, 3,5 μl of MgCl2, 0.2 μl of Taq polymerase (Dupla-Taq™, obtained from ZenonBio Ltd., Szeged, Hungary), add bidistilled water to reach a final volume of 21,2 μl.
The PCR parameters for detecting the pathogens with primers based on the ITS region and with the whole- Candida primers are: preliminary denaturation step at 94°C for 30 seconds, 5 cycles of denaturation step at 94°C for 10 seconds, annealing/extension at 59°C for 20 seconds, 34 cycles of denaturation at 94°C for 10 seconds, annealing/extension at 610C for 17 seconds.
The PCR parameters for detecting the pathogens with primers based on the Phospholipase-D are the following: preliminary denaturation step at 94°C for 30 seconds, 5 cycles of denaturation step at 94°C for 10 seconds, annealing/extension at 59°C for 20 seconds, 34 cycles of denaturation at 94°C for 10 seconds, annealing/extension at 64°C for 17 seconds. Gel electrophoresis: Prepare a 2% of agarose gel by adding 2 ml of 50-fold concentrated TAE buffer to 2 g of agarose and 98 ml of distilled water. Dissolve the agarose in the buffer by using a microwave oven. Run the gel on 60-80 mV for 20 minutes.
Example 2: Detection after cultivation with DNA extraction from blood samples: Two ml of blood sample from BD Vacutainer® Blood Collection Tubes containing heparin is cultivated for a two days period. The cultivation is carried out in heparin containing BD Vacutainer® tubes with 10 ml of YPD medium supplemented with the required antibiotics and 2 ml of the sample at 37 0C on rotary shaker. After one day of incubation 1 ml of the sample is applied into a heparin free tube with 10 ml of YPD medium containing the required antibiotics and incubated at 37 0C on rotary shaker for additional 24 hours. During this cultivation period the number of cells reaches the detectable amount which is 50-80 cells/PCR sample. On the second day the cell content of the whole sample will be around 500000 CFU. Due to the dilutions the heparin content of the sample decreases to 1.4U/ml which is not able to inhibit the polymerase chain reaction.
DNA extraction protocol: Apply 100 μl of the cultivated sample, 300 μl of 20 % N-lauroylsarcosine sodium salt, 300 μl of 5M ammonium acetate and 1 μl of β-mercaptoethanol into a microcentrifuge tube and vortex it 30 seconds. Incubate the mixture at 70 0C for 15 minutes. After the incubation period vortex the sample again and add 600 μl of benzyl alcohol and 50 μl of benzyl benzoate. Centrifuge the mixture at room temperature at 13000 rpm for 15 minutes and pipet the supernatant into a new centrifuge tube. Precipitate the supernatant with 650 μl of isopropyl alcohol and centrifuge again at 13000 rpm for 10 minutes. Discard the supernatant and wash the pel- let with 650 μl of 70 % ethanol, then centrifuge again at 13000 rpm for 5 minutes. Discard the ethanol and dry the sample in a vacuum drier. Resuspend the DNA in 30 μl of bidistilled water and store at -20 0C.
Detecting the presence of Candida in the sample: The test can be carried out by using the WCsunF and WCsunR primer pairs, which were designed on the basis of the 18S rDNA region.
Determination of eight Candida species using primer pairs based on the ITS region: The detectable species are Candida albicans, C. dubliniensis, C. glabrata, C. guilliermodii, C. krusei, C. lusitaniae, C. parapsilosis and C. tropicalis. The working combinations of the primers for identifying the species are: Canforl and Clusi for Candida lusitaniae, Canforl and Cguil for Candida guilliermondii, Canforl and Cglab for Candida glabrata, Canforl and Ckrus for Candida krusei, Canfor 2 and Cdubl for Candida dubliniensis, Canfor 2 and Ctropi for Candida tropicalis, Canfor 2 and Calbic for Candida albicans, Canfor 2 and Cpara for Candida parapsilosis. Determining eight Candida species using primer pairs based on the Phospholipase-D region. The detectable species are Candida albicans, C. dubliniensis, C. glabrata, C. guilliermodii, C. kefyr, C. lusitaniae, C. parapsilosis and C. tropicalis. The working combinations of the primers for identifying the species are: CparkrD and CparaD for Candida parapsilosis, CparkrD and CkrusD for Candida krusei, CguitrD and CguiD for Candida guilliermondii, CguitrD and CtropD for Candida tropicalis, CglkefD and CglabD for Candida glabrata, CglkefD and CkefD for Candida kefyr, CalbduD and CaIbD for Candida albicans, CalbduD and CdubD for Candida dubliniensis.
PCR: Introduce 1-2 μl of sample to a PCR tube containing 2 μl of buffer, 4 μl of dNTP mix, 3-3 μl of primers, 2 μl of MgCl2, 0.2 μl of Taq polymerase (Dupla-Taq™, obtained from ZenonBio Ltd., Szeged, Hungary), add bidistilled water to reach a final volume of 21,2 μl. The PCR parameters for detecting the pathogens with primers based on the ITS region and with the whole -
Candida primers are: preliminary denaturation step at 94°C for 30 seconds, 5 cycles of denaturation step at 94°C for 10 seconds, annealing/extension at 57°C for 20 seconds, 34 cycles of denaturation at 94°C for 10 seconds, annealing/extension at 62°C for 17 seconds.
The PCR parameters for detecting the pathogens with primers based on the Phospholipase-D are the follow- ing: preliminary denaturation step at 94°C for 30 seconds, 5 cycles of denaturation step at 94°C for 10 seconds, annealing/extension at 59°C for 20 seconds, 34 cycles of denaturation at 94°C for 10 seconds, annealing/extension at 64°C for 17 seconds.
Gel electrophoresis: Prepare a 2% of agarose gel by adding 2 ml of 50-fold concentrated TAE buffer to 2 g of agarose and 98 ml of distilled water. Dissolve the agarose in the buffer by using a microwave oven. Run the gel on 60-80 mV for 20 minutes.
Example 3: Identification after solid state media cultivation
Pick one colony of Candida species from any commercially available solid state media which is suitable for culturing yeast (e.g. rice agar, CHROM-agar etc.). Suspend it in 100 μl distilled water. From this suspension 1 μl can be introduced to a PCR tube as the source of the template DNA.
Determination of eight Candida species using primer pairs based on the ITS region: The detectable species are Candida albicans, C. dubliniensis, C. glabrata, C. guilliermodii, C. krusei, C. lusitaniae, C. parapsilosis and C. tropicalis. The working combinations of the primers for identifying the species are: Canforl and Clusi for Candida lusitaniae, Canforl and Cguil for Candida guilliermondii, Canforl and Cglab for Candida glabrata, Canforl and Ckrus for Candida krusei, Canfor 2 and Cdubl for Candida dubliniensis, Canfor 2 and Ctropi for Candida tropicalis, Canfor 2 and Calbic for Candida albicans, Canfor 2 and Cpara for Candida parapsilosis.
Determining eight Candida species using primer pairs based on the Phospholipase-D region. The detectable species are Candida albicans, C. dubliniensis, C. glabrata, C. guilliermodii, C. kefyr, C. lusitaniae, C. parapsilosis and C. tropicalis. The working combinations of the primers for identifying the species are: CparkrD and CparaD for Candida parapsilosis, CparkrD and CkrusD for Candida krusei, CguitrD and CguiD for Candida guilliermondii, CguitrD and CtropD for Candida tropicalis, CglkefD and CglabD for Candida glabrata, CglkefD and CkefD for Candida kefyr, CalbduD and CaIbD for Candida albicans, CalbduD and CdubD for Candida dubliniensis.
PCR: Introduce 1-2 μl of sample to a PCR tube containing 2 μl of buffer, 4 μl of dNTP mix, 3-3 μl of pri- mers, 2 μl of MgCl2, 0.2 μl of Taq polymerase (Dupla-Taq™, obtained from ZenonBio Ltd., Szeged, Hungary), add bidistilled water to reach a final volume of 21,2 μl.
The PCR parameters for detecting the pathogens with primers based on the ITS region and with the whole - Candida primers are: preliminary denaturation step at 94°C for 30 seconds, 5 cycles of denaturation step at 94°C for 10 seconds, annealing/extension at 57°C for 20 seconds, 34 cycles of denaturation at 94°C for 10 seconds, annealing/extension at 62°C for 17 seconds.
The PCR parameters for detecting the pathogens with primers based on the Phospholipase-D are the following: preliminary denaturation step at 94°C for 30 seconds, 5 cycles of denaturation step at 94°C for 10 seconds, annealing/extension at 59°C for 20 seconds, 34 cycles of denaturation at 94°C for 10 seconds, annealing/extension at 64°C for 17 seconds. Gel electrophoresis: Prepare a 2% of agarose gel by adding 2 ml of 50-fold concentrated TAE buffer to 2 g of agarose and 98 ml of distilled water. Dissolve the agarose in the buffer by using a microwave oven. Run the gel on 60-80 mV for 20 minutes.
Example 4: Direct identification from complex human samples excluding blood The source of the samples could be body fluid, nails, skin, sputum, stool, swab of conjunctiva, throat, urine, or vaginal. The samples are cultivated for a two days period. The cultivation is carried out in sterile Erlenmeyer flask or screw cap tube with 10 ml of YPD medium supplemented with the required antibiotics and the sample at 37 0C on rotary shaker. After the incubation period 2 μl of the sample can be applied for direct detection. Detecting the presence of Candida in the sample: The test can be carried out by using the WCsunF and
WCsunR primer pairs, which were designed on the basis of the 18S rDNA region.
Determination of eight Candida species using primer pairs based on the ITS region: The detectable species are Candida albicans, C. dubliniensis, C. glabrata, C. guilliermodii, C. krusei, C. lusitaniae, C. parapsilosis and C. tropicalis. The working combinations of the primers for identifying the species are: Canforl and Clusi for Candida lusitaniae, Canforl and Cguil for Candida guilliermondii, Canforl and Cglab for Candida glabrata, Canforl and Ckrus for Candida krusei, Canfor 2 and Cdubl for Candida dubliniensis, Canfor 2 and Ctropi for Candida tropicalis, Canfor 2 and Calbic for Candida albicans, Canfor 2 and Cpara for Candida parapsilosis.
Determining eight Candida species using primer pairs based on the Phospholipase-D region. The detectable species are Candida albicans, C. dubliniensis, C. glabrata, C. guilliermodii, C. kefyr, C. lusitaniae, C. parapsi- losis and C. tropicalis. The working combinations of the primers for identifying the species are: CparkrD and CparaD for Candida parapsilosis, CparkrD and CkrusD for Candida krusei, CguitrD and CguiD for Candida guilliermondii, CguitrD and CtropD for Candida tropicalis, CglkefD and CglabD for Candida glabrata, CglkefD and CkefD for Candida kefyr, CalbduD and CaIbD for Candida albicans, CalbduD and CdubD for Candida dubliniensis. PCR: Introduce 1-2 μl of sample to a PCR tube containing 2 μl of buffer, 4 μl of dNTP mix, 3-3 μl of primers, 2.5 μl of MgCU, 0.2 μl of Taq polymerase (Dupla-Taq™, obtained from ZenonBio Ltd., Szeged, Hungary), add bidistilled water to reach a final volume of 21,2 μl.
The PCR parameters for detecting the pathogens with primers based on the ITS region and with the whole - Candida primers are: preliminary denaturation step at 94°C for 30 seconds, 5 cycles of denaturation step at 94°C for 10 seconds, annealing/extension at 57°C for 20 seconds, 34 cycles of denaturation at 94°C for 10 seconds, annealing/extension at 62°C for 17 seconds.
The PCR parameters for detecting the pathogens with primers based on the Phospholipase-D are the following: preliminary denaturation step at 94°C for 30 seconds, 5 cycles of denaturation step at 94°C for 10 seconds, annealing/extension at 59°C for 20 seconds, 34 cycles of denaturation at 94°C for 10 seconds, anneal- ing/extension at 64°C for 17 seconds.
Gel electrophoresis: Prepare a 2% of agarose gel by adding 2 ml of 50-fold concentrated TAE buffer to 2 g of agarose and 98 ml of distilled water. Dissolve the agarose in the buffer by using a microwave oven. Run the gel on 60-80 mV for 20 minutes.
Example 5: Detection from complex human samples excluding blood with DNA extraction from the samples
The source of the samples can be body fluid, nails, skin, sputum, stool, swab of conjunctiva, throat, urine, or vaginal. The samples are cultivated for a two days period. The cultivation is carried out in sterile Erlenmeyer flask or screw cap tube with 10 ml of YPD medium supplemented with the required antibiotics and the sample at 37 0C on rotary shaker. DNA extraction protocol: Apply 100 μl of the cultivated sample, 300 μl of 20 % N-lauroylsarcosine sodium salt, 300 μl of 5M ammonium acetate and 1 μl of β-mercaptoethanol into a microcentrifuge tube and vortex it 30 seconds. Incubate the mixture at 70 0C for 15 minutes. After the incubation period vortex the sample again and add 600 μl of benzyl alcohol and 50 μl of benzyl benzoate. Centrifuge the mixture at room temperature at 13000 rpm for 15 minutes and pipet the supernatant into a new centrifuge tube. Precipitate the supernatant with 650 μl of isopropyl alcohol and centrifuge again at 13000 rpm for 10 minutes. Discard the supernatant and wash the pellet with 650 μl of 70 % ethanol, then centrifuge again at 13000 rpm for 5 minutes. Discard the ethanol and dry the sample in a vacuum drier. Resuspend the DNA in 30 μl of bidistilled water and store at -20 0C.
Detecting the presence of Candida in the sample: The test can be carried out by using the WCsunF and WCsunR primer pairs, which were designed on the basis of the 18S rDNA region.
Determination of eight Candida species using primer pairs based on the ITS region: The detectable species are Candida albicans, C. dubliniensis, C. glabrata, C. guilliermodii, C. krusei, C. lusitaniae, C. parapsilosis and C. tropicalis. The working combinations of the primers for identifying the species are: Canforl and Clusi for Candida lusitaniae, Canforl and Cguil for Candida guilliermondii, Canforl and Cglab for Candida glabrata, Canforl and Ckrus for Candida krusei, Canfor 2 and Cdubl for Candida dubliniensis, Canfor 2 and Ctropi for Candida tropicalis, Canfor 2 and Calbic for Candida albicans, Canfor 2 and Cpara for Candida parapsilosis.
Determining eight Candida species using primer pairs based on the Phospholipase-D region. The detectable species are Candida albicans, C. dubliniensis, C. glabrata, C. guilliermondii, C. kefyr, C. lusitaniae, C. parapsilosis and C. tropicalis. The working combinations of the primers for identifying the species are: CparkrD and CparaD for Candida parapsilosis, CparkrD and CkrusD for Candida krusei, CguitrD and CguiD for Candida guilliermondii, CguitrD and CtropD for Candida tropicalis, CglkefD and CglabD for Candida glabrata, CglkefD and CkefD for Candida kefyr, CalbduD and CaIbD for Candida albicans, CalbduD and CdubD for Candida dubliniensis.
PCR: Introduce 1-2 μl of sample to a PCR tube containing 2 μl of buffer, 4 μl of dNTP mix, 3-3 μl of pri- mers, 2 μl of MgCl2, 0.2 μl of Taq polymerase (Dupla-Taq™, obtained from ZenonBio Ltd., Szeged, Hungary), add bidistilled water to reach a final volume of 21,2 μl.
The PCR parameters for detecting the pathogens with primers based on the ITS region and with the whole - Candida primers are: preliminary denaturation step at 94°C for 30 seconds, 5 cycles of denaturation step at 94°C for 10 seconds, annealing/extension at 57°C for 20 seconds, 34 cycles of denaturation at 94°C for 10 seconds, annealing/extension at 62°C for 17 seconds.
The PCR parameters for detecting the pathogens with primers based on the Phospholipase-D are the following: preliminary denaturation step at 94°C for 30 seconds, 5 cycles of denaturation step at 94°C for 10 seconds, annealing/extension at 59°C for 20 seconds, 34 cycles of denaturation at 94°C for 10 seconds, annealing/extension at 64°C for 17 seconds. Gel electrophoresis: Prepare a 2% of agarose gel by adding 2 ml of 50-fold concentrated TAE buffer to 2 g of agarose and 98 ml of distilled water. Dissolve the agarose in the buffer by using a microwave oven. Run the gel on 60-80 mV for 20 minutes.
Example 6: Multiplex Polymerase Chain Reaction The multiplex PCR can be performed directly from the cultivated samples, or by using the DNA extract. Working combinations of the primers for the ITS based reactions: The primer Canforl can be combined in one reaction with C. glabrata (Cglab) and C. lusitaniae (Clusi) specific primers, or with the C. guilliermondii
(Cguil) and C. krusei (Ckrus) specific primers. The primer Canfor2 can be combined in the same PCR tube with
C. albicans (Calbic ) and C. dubliniensis (Cdubl) specific primers, or with the C. tropicalis (Ctropi) and C. pa- rapsilosis (Cpara) specific primers.
The working combinations of the primers designed on the basis of the Phospholipase-D region for the multiplex PCR are: CparkrD with the Candida parapsilosis (CparaD) and Candida krusei (CkrusD) specific primers. CguitrD with the Candida guilliermondii (CguiD) and Candida tropicalis (CtropD) specific primers. CglkefD with the Candida glabrata (CglabD) and Candida kefyr (CkefD) specific primers. CalbduD with the Candida albicans (CaIbD) and Candida dubliniensis (Cdub) specific primers.
PCR for ITS based primers: Introduce 2 μl of sample to a PCR tube containing 2 μl of buffer, 4 μl of dNTP mix, 3 μl of the shared (Canforl or Canfor2) primers, 2-2 μl of the species specific primers, 2.4 μl of MgCl2, 0.2 μl of Taq polymerase (Dupla-Taq™, obtained from ZenonBio Ltd., Szeged, Hungary), add bidistilled water to reach a final volume of 21,2 μl. The PCR parameters for detecting the pathogens with primers based on the ITS region and with the whole -
Candida primers are: preliminary denaturation step at 94°C for 30 seconds, 5 cycles of denaturation step at 94°C for 10 seconds, annealing/extension at 57°C for 20 seconds, 34 cycles of denaturation at 94°C for 10 seconds, annealing/extension at 600C for 17 seconds.
PCR for Phospholipase-D based primers: Introduce 1-2 μl of sample to a PCR tube containing 2 μl of buffer, 4 μl of dNTP mix, 4 μl of the shared (CparkrD, CguitrD, CglkefD, CalbduD) primers, 2-2 μl of the species specific primers, 2.6 μl Of MgCl2, 2 0.2 μl of Taq polymerase (Dupla-Taq™, obtained from ZenonBio Ltd., Szeged, Hungary), add bidistilled water to reach a final volume of 21,2 μl.
The PCR parameters for detecting the pathogens with primers based on the Phospholipase-D are the following: preliminary denaturation step at 94°C for 30 seconds, 5 cycles of denaturation step at 94°C for 10 seconds, annealing/extension at 58°C for 20 seconds, 34 cycles of denaturation at 94°C for 10 seconds, annealing/extension at 64°C for 17 seconds.
Materials and methods applied Cultivation of the samples: Materials:
Antibiotics: Neomycin stock solution (10mg/ml)
Chloramphenicol stock solution (34mg/ml)
For the effective inhibition of the bacterial growth, the final concentration of the antibiotics in the case of Neomycin must be in the range of 10-80 μl/ml and for Chloramphenicol in the range of 0,7-5μl/ml. Fermentation broth (YPD):
For a final volume of 1000 ml
0.5% yeast extract (5g)
1% dextrose (1 Og)
0.5% peptone (5g) DNA extraction protocol:
The extraction is carried out in a 1.5 ml microcentrifuge tubes. Materials: 100 μl of sample 300 μl of 20 % N-lauroylsarcosine sodium salt
300 μl of 5M ammonium acetate
1 μl of β-mercaptoethanol
The mixture is vortex-mixed for 30 second, then incubated at 70 0C for 15 minutes.
After the incubation period the sample is vortex-mixed again The following step is the addition of 600 μl of benzyl alcohol and 50 μl of benzyl benzoate.
The mixture is centrifuged at room temperature at 13000 rpm for 15 minutes and the supernatant is pipetted into a new centrifuge tube. The supernatant is precipitated with 650 μl of isopropyl alcohol and centrifuged again at 13000 rpm for 10 minutes. The supernatant is discarded and the pellet is washed with 650 μl of 70 % ethanol, then centrifuged again at 13000 rpm for 5 minutes. Ethanol is discarded and the sample is dried in a vacuum dryer. The DNA is resuspended in 30 μl of bidistilled water and stored at -20 0C.
PCR protocol:
Materials: Stock solutions: dNTP mix: 960 μl of bidistilled water and 10-10 μl of 100 mM dATP, dCTP, dGTP and dTTP
Primers: 1000-fold dilution with bidistilled water of a lnmol/μl stock The final volume of the reaction is 21.2 μl
2 μl of puffer
4 μl of dNTP mix 4-4 μl of primers
1.6-3.5 μl OfMgCl2
0.2 μl of Taq
1-2 μl of DNA template
Multiplex PCR:
The final volume of the reaction is 21.2 μl 2 μl of puffer 4 μl of dNTP mix 4-2-2 μl of primers 1,6-3.5 μl OfMgCl2
0.2 μl of Taq 1-2 μl of DNA template
Protocol for direct PCR from blood samples: The final volume of the reaction is 21.2 μl 2 μl of puffer 4 μl ofdNTP mix 3-3 μl of primers 3.4-4.7 μl OfMgCl2 0.2 μl of Taq
1-2 μl of DNA template
PCR program for the ITS based reaction:
1. 94°C 30 sec
2. 94°C 10 sec
3. 57-59 0C 20 sec go to step 2 for 5 cycles
4. 94°C 10 sec
5. 60-62 0C 17 sec go to step 4 for 34 cycles
6. 100C CO
PCR program for the Phospholipase-D based reaction:
1. 94°C 30 sec
2. 94°C 10 sec
3. 58-61°C 20 sec go to step 2 for 5 cycles
4. 94°C 10 sec
5. 63-66°C 17 sec go to step 4 for 34 cycles
6. 100C CO
Gel electrophoresis:
Materials:
TAE buffer
1000 ml 50-fold concentrated
242 g of TRIS
57.1 ml of acetic acid 100 ml of 0.5 M EDTA
Ethidium bromide: 5 μl for 100ml of TAE buffer from a 10 mg/ml stock solution
Gel preparation:
Add 2 ml of 50-fold concentrated TAE buffer to 2 g of agarose and 98 ml of distilled water.
Dissolve the agarose in the buffer by using a microwave oven. The electrophoresis can be carried out on 60- 80 mV for 20 minutes.
Sequences of the PCR primers of the invention based on the ITS region:
Canfor 1 shared forward: 5 ' - CTCTTGGTTCTCGC ATC - 3 ' SEQ ID NO: 1
Clusi reverse: 5 ' - TCGAGG AATGCCTCGA - 3 ' SEQ ID NO: 2 Cguil reverse: 5 ' - AG AAAT ATCCCGCC AC A - 3 ' SEQ ID NO: 3 Cglab reverse: 5' - GATTAATAGAGAAGCTTGCG - S ' SEQ ID NO: 4
Ckrus reverse: 5 ' - G ACGCTCTTT AC ACGTCGT - 3 ' SEQ ID NO: 5
Canfor 2 shared forward: 5 ' - ACCTGCGG AAGG ATC ATTA - 3 ' SEQ ID NO: 6
Cdubl reverse: 5 ' - C AAC ACC AAACCCT AGG - 3 ' SEQ ID NO: 7
Ctropi reverse: 5 ' - CGCTT AAAAT AAGTTTCCAC - 3 ' SEQ ID NO: 8
Calbic reverse: 5 ' - CGTGGT AG ACGTT ACCG - 3 ' SEQ ID NO: 9
Cpara reverse: 5 ' - GGTTG AGTTT AATCTCTGGC A - 3 ' SEQ ID NO: 10
Lengths of the amplicons produced using the shared Canfor 1 and the species specific primers: C. lusitaniae: fragment length 112 bp C. guilliermondii: fragment length 294 bp Candida glabrata: fragment length 250 bp Candida krusei: fragment length 196 bp
Lengths of the amplicons produced using the shared Canfor 2 and the species specific primers:
C. dubliniensis: fragment length 344 bp C. tropicalis: fragment length 398 bp C. albicans: fragment length 454 bp
C. parapsilosis: fragment length 123 bp
Primer sequences of the invention used in the genera specific whole-Candida reaction (18S)
Forward primer: WCsunF 5' - AATTGACGGAAGGGCACCACCAGG - 3' SEQ ID NO: 23
Reverse primer:
WCsunR 5 ' - TCAACGCAAGCTGATGACTTGCGC - 3 ' SEQ ID NO: 24
Fragment length: -480 bp
Sequences of the PCR primers of the invention based on the Phospholipase-D region:
CparkrD shared reverse: 5 ' - GTCGTGCC AAGGC ATCCT - 3 ' SEQ ID NO: 11
CparaD forward: 5 ' - GAAC AACTGTTGCC ACTGACT - 3 ' SEQ ID NO: 12
CkrusD forward: 5 ' - GAC ATGGAG AAGCTGCGGT - 3 ' SEQ ID NO: 13
CguitrD shared forward: 5 ' - CACGATTGGTGGCTTTCTCC - 3 ' SEQ ID NO: 14 CguiD reverse: 5' - GCAACAGTCTGGTCAATGACA - S ' SEQ ID NO: 15
CtropD reverse: 5 ' - ATCCGCTGT ATCAT AACG ACC A - 3 ' SEQ ID NO: 16
CglkefD shared reverse: 5 ' - C ATC ATGCC AAGGC ATTCTAGG - 3 ' SEQ ID NO: 17
CglabD forward: 5 ' - CGGT AGCGTTT ATGGG AGG A - 3 ' SEQ ID NO: 18
CkefD forward: 5 ' - CTGGTCATTG AGCG AAGCTCT - 3 ' SEQ ID NO: 19 CalbduD shared reverse: 5 ' - CCTTCTCT ATGTGCTCG AATG A - 3 ' SEQ ID NO: 20 CdubD forward: 5 ' - C ATGGACTAG ATGGT ACCTGC - 3 ' SEQ ID NO: 21
CaIbD forward: 5 ' - GAGTT AAAG ATTTCCTGG AACTCG - 3 ' SEQ ID NO: 22
Lengths of the amplicons produced using the Phospholipase-D region specific primers: C. parapsilosis: fragment length: 408 bp
C. krusei: fragment length: 156 bp C. tropicalis: fragment length: 342 bp C. guilliermodii: fragment length: 293 bp C. glabrata: fragment length: 210 bp C. kefyr: fragment length: 542 bp
C. albicans: fragment length: 475 bp C. dubliniensis: fragment length: 242 bp
REFERENCES Abbas J., Bodey G. P., Hanna H. A., et al., (2000). Candida krusei Fungemia. An Escalating Serious Infection in Immunocompromised Patients. Archives of Internal Medicine 160, 2659-2664.
Almirante, B., Rodriguez, D., Cuenca-Estrella, M., Almela, M., Sanchez, F., Ayats, J., Alonso-Tarres, C, Rodriguez-Tudela, J. L. & Pahissa. A. (2006). Epidemiology, risk factors, and prognosis of Candida parapsilosis bloodstream infections: case-control population-based surveillance study of patients in 240 Barcelona, Spain, from 2002 to 2003. J Clin Microbiol 44, 1681-1685.
Al-Soud W. A., Radstrom P. (2001). Purification and Characterization of PCR -Inhibitory Components in Blood Cells. Journal of Clinical Microbiology, 39, 485^93
Al-Soud W. A., Jonsson L. J., and Radstrom P. (2000). Identification and Characterization of Immunoglobulin G in Blood as a Major Inhibitor of Diagnostic PCR. Journal of Clinical Microbiology, 38, 345-350 Bassetti, M., Righi, E., Costa, A., Fasce, R., Molinari, M. P., Rosso, R., Pallavicini, F. B. & Viscoli, C.
(2006). Epidemiological trends in nosocomial candidemia in intensive care. BMC Infect Dis 6, 21.
Brandt M. E., Harrison L. H., Pass M., Sofair A. N., Huie S., Li R., Morrison C. J., Warnock D. W., Hajjeh R. A. (2000). Candida dubliniensis Fungemia: the First Four Cases in North America. Emerging Infectious Diseases, 6, 1. Fidel Jr. P. L., Vazquez J. A., Sobel J. D. (1999). Candida glabrata: Review of Epidemiology, Pathogenesis, and Clinical Disease with Comparison to C. albicans. Clinical Microbiology Reviews, 12, 80-96.
Girmenia C, Pizzarelli G., Cristini F., Barchiesi F., Spreghini E., Scalise G. and Martino P. (2006). Candida guilliermondii Fungemia in Patients with Hematologic Malignancies. Journal of Clinical Microbiology, 44, 2458-2464. Kremery, V. & Barnes, A. J. (2002). Non-albicans Candida spp. causing fungaemia: pathogenicity and anti- fungal resistance. J Hosp Infect 50, 243-260.
Levy, L, Rubin, L. G., Vasishtha, S., Tucci, V. & Sood, S. E. (1998). Emergence of 310 Candida parapsilosis as the predominant species causing candidemia in children. Clin Infect Dis 26, 1086-1088. Meis J. F. G. M., Ruhnke M., De Pauw B. E., Odds F. C, Siegert W. and Verweij P. E. (1999). Candida dubliniensis Candidemia in Patients with Chemotherapy -Induced Neutropenia and Bone Marrow Transplantation Emerging Infectious Diseases, 5, 1.
Messer, S. A., Jones, R. N. & Fritsche, T. R. (2006). International surveillance of Candida spp. and Asper- gillus spp.: Report from the SENTRY Antimicrobial Surveillance Program (2003). J Clin Microbiol 44, 1782- 1787.
Peyron F., Favel A., Michel-Nguyen A., Gilly M., Regli P. and Bolmstrόm A. (2001). Improved Detection of Amphotericin B-Resistant Isolates of Candida lusitaniae by Etest. Journal of Clinical Microbiology, 39, 339- 342. Safdar, A., Perlin, D. S. & Armstrong, D. (2002). Hematogenous infections due to Candida parapsilosis: changing trends in fungemic patients at a comprehensive cancer center during the last four decades. Diagn Microbiol Infect Dis 44, 11-16.
Weems, J. J. Jr. (1992). Candida parapsilosis: epidemiology, pathogenicity, clinical manifestations, and antimicrobial susceptibility. Clin Infect Dis 14, 756-766. White P. L., Archer A. E. and Barnes R. A. (2004). Comparison of Non-Culture-Based Methods for Detection of Systemic Fungal Infections, with an Emphasis on Invasive Candida Infections. Journal of Clinical Microbiology 43, 2181-2187
Yokota, M., Tatsumi N., Nathalang O., Yamada T., Tsuda I. (1999). Effects of heparin on polymerase chain reaction for blood white cells. Journal of Clinical Laboratory Analysis 13, 133-140.
SEQUENCE LISTING
<110> Szerodiagnosztikum Kft.
<120> PCR based diagnostic method for the identification of clinically important Candida species
<130> 103897
<160> 24
<170> Patentln version 3.5
<210> 1 <211> 17
<212> DNA
<213> Candida species
<400> 1 ctcttggttc tcgcatc 17
<210> 2
<211> 16
<212> DNA
<213> Candida lusitaniae
<400> 2 ttccggaaggggaaaattgg ccccttccggaa 16
<210> 3 <211> 17
<212> DNA
<213> Candida guilliermondii
<400> 3 agaaatatcc cgccaca 17
<210> 4
<211> 20
<212> DNA
<213> Candida glabrata <400> 4 gattaataga gaagcttgcg 20
<210> 5
<211> 19
<212> DNA <213> Candida krusei
<400> 5 gacgctcttt acacgtcgt 19
<210> 6
<211> 19 <212> DNA
<213> Candida species
<400> 6 acctgcggaa ggatcatta 19
<210> 7 <211> 17
<212> DNA
<213> Candida dubliniensis
<400> 7 caacaccaaa ccctagg 17 <210> 8
<211> 20
<212> DNA
<213> Candida tropicalis
<400> 8 cgcttaaaat aagtttccac 20
<210> 9
<211> 17
<212> DNA
<213> Candida albicans <400> 9 cgtggtagac gttaccg 17 <210> 10
<211> 21
<212> DNA
<213> Candida parapsilosis <400> 10 ggttgagttt aatctctggc a 21
<210> 11
<211> 18
<212> DNA <213> Candida species
<400> 11 gtcgtgccaa ggcatcct 18
<210> 12
<211> 21 <212> DNA
<213> Candida parapsilosis
<400> 12 gaacaactgt tgccactgac t 21
<210> 13 <211> 19
<212> DNA
<213> Candida krusei
<400> 13 gacatggaga agctgcggt 19 <210> 14
<211> 20
<212> DNA
<213> Candida species
<400> 14 cacgattggt ggctttctcc 20
<210> 15
<211> 21
<212> DNA
<213> Candida guilliermondii <400> 15 gcaacagtct ggtcaatgac a 21
<210> 16
<211> 22
<212> DNA <213> Candida tropicalis
<400> 16 atccgctgta tcataacgac ca 22
<210> 17
<211> 22
<212> DNA
<213> Candida species
<400> 17 catcatgcca aggcattcta gg 22
<210> 18
<211> 20
<212> DNA
<213> Candida glabrata
<400> 18 cggtagcgtt tatgggagga 20
<210> 19
<211> 21
<212> DNA
<213> Candida kefyr
<400> 19 ctggtcattg agcgaagctc t 21
<210> 20
<211> 22
<212> DNA
<213> Candida species
<400> 20 ccccttttccttccttaatt ggttggccttccggaaaatt ggaa 22
<210> 21
<211> 21
<212> DNA
<213> Candida dubliniensis
<400> 21 catggactag atggtacctg c 21
<210> 22
<211> 24
<212> DNA
<213> Candida albicans
<400> 22 gagttaaaga tttcctggaa ctcg 24
<210> 23
<211> 24
<212> DNA
<213> Candida species <400> 23 aattgacgga agggcaccac cagg 24
<210> 24
<211> 24
<212> DNA
<213> Candida species
<400> 24 tcaacgcaag ctgatgactt gcgc 24

Claims

I. A PCR based method for detecting the presence of at least one pathogenic Candida species present in a heparin containing blood sample, wherein a part of said heparin containing blood sample, comprising material serving as PCR template, is directly added to a Candida species specific PCR reaction mixture.
2. A method according to Claim 1, wherein said Candida species specific PCR reaction mixture comprises a non-heparin resistant Taq DNA polymerase.
3. A method according to any preceding claim, wherein said blood sample, prior to the addition of said part thereof to said PCR reaction mixture, is preincubated, advantageously for 1 -3 days, in conditions facilitating the growth of said at least one pathogenic Candida species being supposedly present in said blood sample.
4. A method according to any preceding claim, wherein said blood sample is preincubated in the presence of at least one antibiotic, advantageously Neomycin and/or Chloramphenicol.
5. A method according to any preceding claim, wherein said PCR reaction mixture contains less than 0.06 IU heparin, advantageously less than 0.05 IU heparin.
6. A method according to any preceding claim, wherein said PCR reaction mixture has a magnesium ion concentration of at least 4 mM, advantageously at least 5 mM.
7. A method according to any preceding claim, wherein said at least one pathogenic Candida species is selected from the group consisting of C. glabrata, C. lusitaniae, C. guilliermondii, C. krusei, C. albicans, C. dubli- niensis, C. tropicalis, C. parapsilosis and C. kefyr.
8. A method according to any preceding claim, wherein said at least one pathogenic Candida species is identified on the basis of the size of the amplicon produced in said Candida species specific PCR reaction mixture.
9. A method according to any preceding claim, wherein the Candida species specific PCR primer pair(s) comprised in said Candida species specific PCR reaction mixture is (are) designed to be capable of specifically hybridizing either to a sequence region present in the internal transcribed spacer (ITS) region of the ribosomal gene repeat or to a species specific sequence region present in the Phospholipase-D gene of said at least one pathogenic Candida species.
10. A method according to any preceding claim, wherein the sequences of said Candida species specific PCR primer pairs are selected from the group consisting of sequences identified by any of SEQ ID NOs from 1 to 22, sequences at least 90% identical with sequences identified by any of SEQ ID NOs from 1 to 22, and com- plementary sequences thereof.
II. A PCR based method for detecting the presence of at least one pathogenic Candida species present in a biological sample, wherein the sequences of the applied Candida species specific PCR primer pairs are selected from the group consisting of sequences identified by any of SEQ ID NOs from 1 to 22, sequences being at least 90% identical with sequences identified by any of SEQ ID NOs from 1 to 22, and complementary sequences thereof.
12. A method according to any preceding claims, wherein said at least one pathogenic Candida species is selected from the group consisting of C. glabrata, C. lusitaniae, C. guilliermondii, C. krusei, C. albicans, C. dubliniensis, C. tropicalis, C. parapsilosis and C. kefyr.
13. A method according to any preceding claim, wherein said Candida species specific PCR reaction mixture comprises more than one, advantageously more than two PCR primer pairs being specific for more than one, advantageously more than two Candida species, and wherein said primers, upon suitable thermal cycling, enable the production of more than one, advantageously more than two species specific amplicons each having a characteristic individual size being gel-electrophoretically distinguishable from any other Candida species specific amplicon that may be produced in said Candida species specific PCR reaction mixture.
14. A method according to any preceding claim, wherein at least one of the individual PCR primers being present in said Candida species specific PCR reaction mixture is capable of specifically hybridizing to target sequences originating from more than one, advantageously more than two different Candida species being supposedly present in said blood or other biological sample.
15. A method according to any preceding claim for detecting the presence of two pathogenic Candida species selected from the group consisting of C. glabrata, C. lusitaniae, C. guilliermondii, C. krusei, C. albicans, C. dubliniensis, C. tropicalis, C. parapsilosis and C. kefyr in one single Candida species specific PCR reaction, wherein said primers applied in said one single Candida species specific PCR reaction have the sequences identified by a) SEQ ID NO: 1, SEQ ID NO: 2 and SEQ ID NO: 4 for C. glabrata and C. lusitaniae; or b) SEQ ID NO: 1, SEQ ID NO: 3 and SEQ ID NO: 5 for C. guilliermondii and C. krusei; or c) SEQ ID NO: 6, SEQ ID NO: 7 and SEQ ID NO: 9 for C. albicans and C. dubliniensis; or d) SEQ ID NO: 6, SEQ ID NO: 8 and SEQ ID NO: 10 for C. tropicalis and C. parapsilosis; or e) SEQ ID NO: 11, SEQ ID NO: 12 and SEQ ID NO: 13 for C. parapsilosis and C. krusei; or f) SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16 for C. guilliermondii and C. tropicalis; or g) SEQ ID NO: 17, SEQ ID NO: 18 and SEQ ID NO: 19 for C. glabrata and C. kefyr; or h) SEQ ID NO: 20, SEQ ID NO: 21 and SEQ ID NO: 22 for C. dubliniensis and C. albicans; or sequences being at least 90% identical with sequences defined in the above paragraphs a)-h) or complementary sequences thereof, and wherein the identification of said two pathogenic Candida species is advantageously done based on the sizes of their species specific amplicons produced in said single species specific PCR reaction, advantageously determined by gel electrophoresis.
16. A PCR based method for detecting the presence of at least one pathogenic Candida species present in a biological sample, wherein the Candida species specific PCR primer pair(s) applied is(are) designed to be capable of specifically hybridizing to a species specific sequence region present in the Phospholipase-D gene of said at least one pathogenic Candida species.
17. A method according to claim 16, wherein said Candida species specific PCR primer pair(s) applied are selected from the group consisting of sequences identified by any of SEQ ID NOs from 11 to 22, sequences at least 90% identical with sequences identified by any of SEQ ID NOs from 11 to 22, and complementary sequences thereof.
18. A reagent kit for detecting the presence of at least one pathogenic Candida species selected from the group consisting of C. glabrata, C. lusitaniae, C. guilliermondii, C. krusei, C. albicans, C. dubliniensis, C. tropi- calis, C. parapsilosis and C. kefyr by a PCR based method, comprising at least one primer pair wherein each primer having a sequence selected from the group consisting of sequences identified by any of SEQ ID NOs from 1 to 22, sequences being at least 90% identical with sequences identified by any of SEQ ID NOs from 1 to 22, and complementary sequences thereof and optionally other reagents suitable for facilitating PCR reactions.
19. A reagent kit for detecting the presence of at least two pathogenic Candida species selected from the group consisting of C. glabrata, C. lusitaniae, C. guilliermondii, C. krusei, C. albicans, C. dubliniensis, C. tropi- calis, C. parapsilosis and C. kefyr by a PCR based method, comprising at least 3 primers having a sequence selected from the group consisting of sequences as defined in any of paragraphs a)-h) of claim 15, sequences being at least 90% identical with sequences defined in said paragraphs or complementary sequences thereof, and optionally other reagents suitable for facilitating PCR reactions.
20. A reagent kit for genera specific detection of the presence of a Candida species, said species being advantageously selected from the group consisting of C. glabrata, C. lusitaniae, C. guilliermondii, C. krusei, C. albicans, C. dubliniensis, C. tropicalis, C. parapsilosis and C. kefyr by a PCR based method, comprising an oligonucleotide primer pair having sequences selected from the group consisting of sequences identified by SEQ ID NOs 23 and 24, respectively, sequences being at least 90% identical with sequences identified by SEQ ID NOs 23 and 24, respectively, and complementary sequences thereof, and optionally other reagents suitable for facilitating PCR reactions.
21. Use of an oligonucleotide having a sequence selected from the group consisting of sequences identified by any of SEQ ID NOs from 1 to 22, sequences being at least 90% identical with sequences identified by any of SEQ ID NOs from 1 to 22 and complementary sequences thereof for detecting, by a PCR based method, the presence of at least one pathogenic Candida species selected from the group consisting of C. glabrata, C. lusitaniae, C. guilliermondii, C. krusei, C. albicans, C. dubliniensis, C. tropicalis, C. parapsilosis and C. kefyr in a biological sample.
22. Use of an oligonucleotide having a sequence selected from the group consisting of sequences identified by any of SEQ ID NOs from 11 to 22, sequences being at least 90% identical with sequences identified by any of SEQ ID NOs from 11 to 22 and complementary sequences thereof for detecting the presence of at least one pathogenic Candida species selected from the group consisting of C. glabrata, C. guilliermondii, C. krusei, C. albicans, C. dubliniensis, C. tropicalis, C. parapsilosis and C. kefyr in a biological sample.
23. Use of a primer oligonucleotide for detecting, by a PCR based method, the presence of a pathogenic Candida species selected from the group consisting of C. glabrata, C. lusitaniae, C. guilliermondii, C. krusei, C. albicans, C. dubliniensis, C. tropicalis, C. parapsilosis and C. kefyr in a heparin containing blood sample, wherein a part of said heparin containing blood sample is directly added to the PCR reaction mixture.
24. The use according to Claim 23, wherein said PCR reaction mixture comprises a non-heparin resistant Taq DNA polymerase.
25. Use of a primer oligonucleotide for detecting, by a PCR based method, the presence of a pathogenic Candida species selected from the group consisting of C. glabrata, C. guilliermondii, C. krusei, C. albicans, C. dubliniensis, C. tropicalis, C. parapsilosis and C. kefyr, wherein said oligonucleotide has a sequence designed to be capable of specifically hybridizing to a species specific sequence region present in the Phospholipase-D gene of said pathogenic Candida species.
26. Use of an oligonucleotide primer pair having sequences selected from the group consisting of sequences identified by SEQ ID NOs 23 and 24, respectively, sequences being at least 90% identical with sequences identified by SEQ ID NOs 23 and 24, respectively, and complementary sequences thereof for genera specific detection, by a PCR based method, the presence of a Candida species, said species being advantageously selected from the group consisting of C. glabrata, C. lusitaniae, C. guilliermondii, C. krusei, C. albicans, C. dubliniensis, C. tropicalis, C. parapsilosis and C. kefyr in a biological sample.
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