HK1206396B - A novel method for simultaneous detection and discrimination of bacterial, fungal, parasitic and viral infections of eye and central nervous system - Google Patents

Description

Novel method for simultaneously detecting and identifying bacterial, fungal, parasitic and viral infections of the eye and central nervous system

The present application is a divisional application of invention patent application No. 200880000035.8 having application date of 27/05/2008 and entitled "novel method for simultaneously detecting and identifying bacterial, fungal, parasitic and viral infections of the eye and central nervous system".

Technical Field

The present invention relates to diagnostic methods for identifying the causative single pathogen or more than one pathogen of ocular and central system infections among many possible pathogens capable of causing the infections. All pathogens affecting individual areas of the eye or central system generally cause the same manifestations or symptoms. The present invention relates to the detection and identification of this pathogen among this set of possible pathogens in a single test without resorting to a kit of tests for one detection of one pathogen. The present invention is directed to symptom-based diagnosis as an alternative to diagnosis based on detection of individual pathogens.

Background

Infections of the eye can be clinically classified according to the anatomical classification that is loaded with the infectious agent and subsequently affected by the infection. There are many organisms that can cause eye infections, and they are described in "Principles and Practice of infectious diseases, 6^thEdition, Gerald Mandell et al (Eds) Elsevier Churchill Livingston, pp1387-1418(2005) ", the disclosure of which is incorporated herein by reference.

Eye infections can be classified into the following categories based on initial clinical diagnosis:

1. infections of the outer eye, e.g. keratitis and conjunctivitis

2. Infectious endophthalmitis

3. Uveitis

4. Retinitis

Many external eye infections are caused by several bacteria and fungi. In fact, the fact that conjunctiva and cornea carry many non-pathogenic bacteria and fungi as parasites due to exposure to the environment is detrimental to the detection of specific pathogens (bacteria and fungi) extracted from conjunctiva and corneal shavings or swabs. In the presence of suppuration or ulcers with pus, physicians make a provisional diagnosis of bacterial infections (proviral diagnosis) and treat the patients with broad-spectrum antibiotics for topical use. However, important infections that are difficult to diagnose but particularly curable are:

● herpes simplex (causing keratitis)

● adenovirus conjunctiva-keratitis (sometimes causing epidemic spread)

● Chlamydia trachomatis (causing follicular conjunctivitis and adult inclusion body conjunctivitis which cause trachoma)

● varicella conjunctivitis (also known as herpes zoster conjunctivitis)

● fast growing mycobacteria such as M.chelonae and M.fortuitum (causing infection after LASIK surgery for reducing ametropia)

Infectious endophthalmitis may be commonly caused by the following pathogens:

● gram-positive bacterium

● gram-negative bacteria

● anaerobic organism, Propionibacterium acnes

● fungi.

It is very common that infection occurs and spreads very quickly after surgery, resulting in blindness. The most important information needed for treatment is whether the pathogen is a bacterium or a fungus, and if it is a bacterium, it is aerobic or anaerobic. Endogenous infections caused by blood borne transmission are rare.

Uveitis is commonly caused by the following pathogens:

● Mycobacterium tuberculosis (Mycobacterium tuberculosis)

● Mycobacterium chelonae (M. chelonae)

● Mycobacterium fortuitum (M.fortuitum)

● Toxoplasma gondii (Toxoplasma gondii).

Retinitis is commonly seen in immunosuppressed individuals and is caused by the following pathogens:

● Cytomegalovirus

● herpes simplex virus

● varicella zoster virus

Severe vision loss occurs in all these patients, and thus early and timely diagnosis of these organisms is important to prevent blindness throughout the eye. The actual incidence of these infections may be relatively high in developing countries. Many diagnostic techniques are used for the diagnosis of eye infections as detailed in the prior art section.

Central nervous system infections can be divided into the following categories:

acute suppurative meningitis: are generally found in children and are caused by the organisms listed below:

● Haemophilus influenzae (Haemophilus influenza)

● Neisseria meningitidis (Neisseria meningitidis) and

● Streptococcus pneumoniae (Streptococcus pneumoniae)

Bacterial culture or smear microscopy of cerebrospinal fluid (CSF) lacks sensitivity. An additional complicating factor is that prior treatment of patients with antibiotics may lead to false negative results in both gram staining and incubation of CSF. For these reasons, physicians are reluctant to trust the culture results and tend to complete a 10-14 day complete course of intravenous antibiotics, which is in most cases unnecessary. Once partially treated, these conditions cannot be distinguished from chronic meningitis caused by the following pathogens:

● Mycobacterium tuberculosis (Mycobacterium tuberculosis)

● various fungi

● is caused by a series of viral encephalitis that can be controlled by treatment, such as HSV, CMV and VZV.

Encephalitis is usually caused by a wide variety of viruses, both endemic and epidemic. However, herpes simplex, cytomegalovirus and varicella zoster are viruses with specific antiviral therapies. Other pathogens that can treat encephalitis are Toxoplasma gondii (Toxoplasma gondii).

Prior Art

Classical methods for detecting pathogens (bacteria, yeast and other fungi, parasites and viruses) in clinical samples involve incubating the sample to expand the number of pathogens present until observable colony growth, which can then be identified and counted by standard laboratory tests. The culture may also be subjected to additional tests to determine the susceptibility of the pathogen to drug treatment, if desired. In order to accurately identify the type of infection, the physician must rely on culture results that may require anywhere from 3 days (as in the case of most bacteria, including fast growing mycobacteria) to 8 weeks (as in the case of tubercle bacilli). This culture is then followed by extensive biochemical tests that may take additional days or even weeks to accurately identify the species of bacteria. In most cases, it is important that this determination be done quickly due to the severity of the disease and the necessity for immediate pharmaceutical intervention. The culture techniques referred to herein are most likely useful in the diagnosis of bacterial and fungal infections, and they are not generally used in the diagnosis of viral infections, particularly since the frequency of virus isolation from clinical specimens by culture is less than 15%.

The appropriate sample for diagnosing infections (e.g., eye infections and nervous system infections) is another key issue for successful diagnosis by detecting the causative agent of a condition (e.g., keratoconjunctivitis, endophthalmitis, uveitis, meningitis, and encephalitis). In these cases, the infection is highly localized and is therefore localized to the eye or CNS. Body fluids, such as blood, plasma or serum, do not contain infectious agents. Infections of the outer eye require samples such as corneal shavings or conjunctival swabs, whereas infectious endophthalmitis require either a vitrectomy performed in an ophthalmic clinic or better a vitrectomy sample, in which case 20ml of a vitreous wash of hank's balanced salt solution is removed in the operating room. Simple vitreous aspirates are often insufficient to diagnose fungal and bacterial endophthalmitis by smear examination and culture. In both cases of uveitis and retinitis, the better sample is 0.2-0.3mL of vitreous humor collected in a 27 gauge needle. The best biological fluid for diagnosing central nervous system infections is cerebrospinal fluid. In one embodiment of the invention, aqueous humor or vitreous humor is sufficient to detect and identify infectious agents in the case of endophthalmitis. This eliminates the necessity of performing a surgical step (such as vitrectomy) in the operating room.

The DNA-based method of identifying pathogens provides a simple, robust and reliable alternative to classical methods that are time consuming and require personnel with specialized training and skill. In classical methods, errors can occur which sometimes lead to an undefined identification of the pathogen and thus to an incorrect diagnosis/treatment. On the other hand, DNA-based pathogen identification has the advantage of identifying the pathogen at a much earlier stage, sometimes earlier than the clinical symptoms observed (subclinical stage). Once the conditions are standardized, the identification of the pathogen is very simple and reliable and can be done by semi-skilled personnel. DNA-based methods can also be used to assess the outcome of a medical intervention (prognostic value). Screening clinical specimens of human pathogens using DNA-based methods such as PCR provides a sensitive and definitive diagnosis and the onset of effective therapy, even for small amounts of clinical specimens (aqueous humor, vitreous humor, tear fluid, saliva, blood, cerebrospinal fluid, mucosal or epithelial scrapings such as corneal scrapings, conjunctival swabs, tissue samples, etc.) that contain very few (about 20-50) pathogenic organisms per specimen.

A potential benefit of using Polymerase Chain Reaction (PCR) technology is the identification of specific bacterial or viral pathogens in a relatively short period of time. The available PCR-based tests have the ability to help the physician decide how to start the treatment while the patient is still in the emergency room. Since PCR-based detection methods do not rely on the presence of living organisms, but on genetic material, PCR-based techniques can be applied to all patient situations, even when antibiotics are used prior to the extraction and collection of clinical specimens. However, the PCR-based method has some difficulties such as false positive results due to contaminated nucleic acids and inhibition of PCR reaction due to complicated samples. The following PCR assays have been used for organisms causing infections of the eye and CNS.

Herpes simplex 1&2: PCR-based DNA detection of HSV has been shown to have 4-5 fold higher sensitivity than virus culture and is not sensitive to transport condition (transport condition), as described in Wald a, et al, "Polymerase chain reaction for detection of bacterial geometry virus (HSV) on multiplex surface: comparison with HSV isolation in cell culture ". j.inf.dis.188: 1345 and 1351 (2003). PCR has been used to detect HSV (Madhavan HN et al, "Detection of Human Simple Virus (HSV) using polymeric reaction (PCR) in ocular samples (such as aqueous humor, corneal scrapings, lens aspirates, lens pocket material and vitreous humor) and other clinical samples (such as CSF, genital tract swabs and cervical swabs) J.Clin.Virol.14: 145. 151 (1999)). Here, PCR enables the detection of 1-3 HSV particles in one clinical sample. PCR is also effectively used to identify herpes virus serotypes, as one of the inventors mentions in published articles, by combining PCR with restriction length polymorphisms of amplicons (Madhavan HN et al, "nutritional and genomic methods for the detection of viral simple virus sera". J.Virol. methods, 108: 97-102(2003)), the disclosure of which is incorporated herein by reference.

Varicella zoster virus:PCR has been used to detect varicella infection (Burke DG, et al, "polymeric infection detection and clinical diagnosis of varicella zone antibacterial infection". J.Inf.Dis, 176: 1080(1996)). Detection of both HSV and VZV in the central nervous system was also achieved by using PCR (Sauerbrei a and Wutzler p. "Laboratory diagnosis of central nervous system treated by human viruses". j.clin. virol, 25s45-s51, (2002)), from which the authors concluded that PCR is the gold standard for detection of VZV, the disclosure of which is incorporated herein by reference.

Cytomegalovirus A: a commercially available PCR assay kit called COBASS Amplicor CMV Monitor of Roche molecular diagnostics (Pleasanton, CA, USA) utilizes the 365 base pair region of the DNA polymerase gene of CMV for amplification detection. Many Detection methods utilize genes for early burst antigens and DNA polymerases to detect the presence of CMV in various body fluids (Stanier P, et al, "Detection of human cytokine induced single cells and urine samples using PCR". mol.cell Probes, 6: 51-58(1992) and Gerna G, et al, "Monitoring human cytokine induced infection and gated clone efficiency in heart transplant transcription side determination of viral emia, anti-gene and DNA emia". j.inf.dis., 164, 488-498(1991), the disclosure of which is incorporated herein by reference). CMV infection in patients is also detected by nested pcr (nested pcr) of various samples, such as blood, amniotic fluid, nasal aspirates, bronchoalveolar lavage fluid, urine, placental material, and bronchial aspirates.

Eye infections caused by all three herpes viruses (HSV, VZV and CMV) were detected by the PCR method as described in the article published by one of the inventors (Priya K et al, "Association of viruses in aqueous humour of pathogens with serogenes: a polymeric chain reaction basedstudy". Ocular immunity and infection 9: 1-9(2003), the disclosure of which is incorporated herein by reference). In this study, nested PCR was performed to detect VZV in order to obtain the necessary sensitivity. However, nested PCR has the concomitant problem of introducing amplicon contamination in the laboratory, since the products of the first round of amplification must be transferred to a second PCR tube containing a second set of primers for amplifying a smaller region of the gene amplified by the first round of PCR.

Detection of Mycobacterium tuberculosis (Mycobacterium tuberculosis) by PCR is a protocol for two FDA-approved tests (see The Amplified Mycobacterium tuberculosis Direct test Gen-Probe, SanDeigo, USA and Amplicor M. tuberculosis test of Roche Diagnostic Systems, Basel, Switzerland). Many other methods of testing are known in the art. However, tuberculosis of the eye has been detected by PCR (Madhavan HN et al, "Polymerase chain reaction for detection of Mycobacterium tuberculosis in epistrical membrane in Eales'". disease. invest. Ophthalmol. Vis. Sci.41: 822-825 (2000)).

Detection of Chlamydia trachomatis by nucleic acid amplification has been used in clinical tests and the method is described in Black CM, "Current methods of laboratory diagnosis of Chlamydia trachomatis infection". clin. microbiol. rev.10: 160-184(1997) in detail. Chlamydial conjunctivitis is detected using PCR on membrane swabs, and as few as 30 organisms are detected in clinical specimens, such as malathi. jet, et al, "aqueous based study on prediction of junctional vision du to chlamydial meningitis", ind.j.med.res, 117: 71-75 (2003).

Adenovirus conjunctivitis was diagnosed by nested PCR of conjunctival swabs as described previously (Dalapath S et al, "Development and use of nested Polymerase Chain Reaction (PCR) for the detection of adenovirus from junctional properties species". J.Clin.Virol.11: 77-84 (1998)).

Toxoplasma gondii causes severe encephalitis and uveitis in patients with immunodeficiency. A number of PCR assay protocols have been used to study various body fluids and tissues, and all PCR assays rely on the amplification of the B1 gene (Danise A, et al, "Use of polymerization reaction assays of aqueous humor in vivo of intracellular diagnosis". Clin. Infect. Dis 24: 1100-1106(1997), monomer. et al, "Use of polymerization reaction in diagnosis of intracellular diagnosis. Ophtology", 106: 1554-1563 (1999)).

Such as "Use of Polymerase Chain Reaction (PCR) and DNA hybridization to a derivative of the interaction of the bacteria inter-microbial fluids of tissues with endorphism", J.Infection, 41: 221-226(2000), infectious endophthalmitis caused by post-operative infection of the eye was studied using PCR reactions of eubacterial genes and by differentiating gram-positive and gram-negative probes. This study was able to detect as few as 6 bacteria in clinical samples. Eye infections caused by anaerobic organisms (such as propionibacterium acnes) such as the disease KL et al, "polymer chain reaction in the diagnosis of bacterial endopthalmitis", brit.j. opthal.82: 1078-1082(1998) using PCR. Such as the "polymerization reaction in the diagnosis of Aspergillus endorhamtis", Ind.J.Med.Res.114: 133-140(2001) and Anand AR et al, "Use of polymerization reaction in fundamental endophathy". Ophthalmology 108: 326-330(2001), fungal endophthalmitis can also be rapidly diagnosed using PCR. In both studies, about 0.4pg of fungal DNA could be detected.

The various PCR assays described herein employ different thermal cycling of the reaction because the primer sets and the genes being detected are different in each PCR. In addition, the reagent concentrations for each of the above-described PCRs were adjusted to optimize the PCR for maximum sensitivity for the set of reactants described herein. The optimal reaction conditions vary depending on the sequence of the nucleotide strand to be amplified, its size and the complexity of the entire target DNA of the organism or pathogen to be detected.

However, there is also a need for a PCR-based assay that can simultaneously detect and identify pathogens that cause bacterial, fungal, parasitic and viral infections of the eye and central nervous system that, in addition to being rapid, is not prone to contamination, and has enhanced sensitivity and specificity over other methods. The method is easy to use in a clinical setting where identification of infectious agents within 24-48 hours is important for life saving. The key problem in accurately diagnosing eye and brain infections can be summarized as:

● infections of the eye and brain are highly localized. There are no traces of pathogens in readily available biological fluids such as blood, serum, plasma, saliva or pus or purulent discharge from external wounds or ulcers.

● any of the well-recognized biomarkers of acute infection, such as C-reactive protein, are common and shared for all infections affecting humans. And thus is non-specific.

● to identify a particular pathogen, samples from the eye or CSF are required. The samples obtained were corneal scrapings for corneal infection, conjunctival swabs for conjunctivitis, aqueous humor for endophthalmitis, and vitreous humor for uveitis and retinitis. In the case of brain infections, the preferred sample is CSF consistently. In all of these samples, there is a limit to the amount of sample that can be obtained from a patient by a single sampling. Typically, you take thousands of cells and perhaps two to three milligrams of sample in a corneal scrapings or swab. The aqueous humor that may be taken from a patient's eye at any time is approximately 100 μ L, and the vitrectomy specimen can reach up to 200 μ L. Up to 5ml CSF samples can be taken, but the volume of CSF sample required for PCR is less than 0.5 ml.

● the limitation in sample volume results in a limitation in the number of bacteria/viruses/parasites present in the sample. In addition, the amount of infectious agents is also lower than that observed in many other body compartments (body components), such as blood.

● the total number of infectious particles present in the sample is directly related to the success of the detection. Below the critical mass, the infectious agents bacteria and fungi cannot grow in the culture medium and are therefore difficult to diagnose. The number of virus particles present in the ocular sample is typically below the detection limit of fluorescent antibody detection experiments and virus culture, thus making the detection sensitivity below 25%. There are no convenient detection systems for parasites such as Toxoplasma gondii (Toxoplasma gondii) and the number of parasites is too small for detection. IgM or IgG assays, such as for HSV, CMV, VZV, adenovirus, Chlamydia and Toxoplasma, are very specific and therefore not diagnostically meaningful.

● another major difficulty in diagnosis is that all of the above-described ocular disorders are acute and require immediate and accurate diagnosis to guide appropriate treatment. Infectious endophthalmitis and necrotizing retinitis caused by viral infection require treatment within 96 hours of the first symptoms, in which case a delay of more than 48 hours will result in blindness.

Multiplex PCR has also been used for certain pathogens in established situations, and the amplification products are identified by determining the molecular weight using mass spectrometry. See Detection and identification of the base composition of PCR products, (Ecker, David J.: Griffey Richard 11.: Sampath, Rangarajan; Hofstandler, Steven A.: Meneil, John; Crooke, Sstanley T. (USA) US patent application publication U.S. Pat.appl.Publ. (2004), pp.168, continuation of the section of US application No. 323,233 US application publication No. US 2003-66220030220030220030220030911 priority: US 2001-79800720010302; US 2002-44431310021206; US 2002-323232323232322002; US 2005132324132411218; US-002301411218; US-4444321218; US 200444444444444430021206; US 2002-323232321218; US 200442003-4432321218).

Gel electrophoresis following multiplex PCR to identify products was attempted for infection of the central nervous system as Read, SJ. and Kurtz, JB. "Laboratory diagnosis of common viral infections of the central nervous system by using a single multiplex PCR screening assay" J.Clin.Microbiol.37: 1352-1355 (1999).

The multiplex PCR is followed by microarrays for detection of pathogens reportedly for detection of pathogens causing respiratory diseases (Wang D et al, "Microarray based detection and genotyping of viral pathogens". Proc. Nat. Acad. Sci. USA 99: 15687-. Detection of amplicons has also attempted using a colorimetric microtiter plate assay system in which the amplicons are labeled with digoxigenin 11-dUTP and biotinylated probes are used to capture the amplicons on a titer plate. The product was displayed using an enzyme-labeled anti-digoxin (Smalling TW et al, "Molecular aproacids to detecting peptides simple virus and antibodies in central nervous system". J.Clin. Microbiol.40: 2317-2322 (2002)).

Such as Madhavan HN et al, "Development and application of a novel polymerase chain reaction for a differentiation of a human cytomegavirus in a clinical specimen", J Virol methods.141: 166-72(2007), multiplex PCR assays were successfully attempted to quantify the virus in clinical samples for three different genes of the same organism, namely the cytomegalovirus morphotropic region II, UL83 and the glycoprotein O gene.

After amplification of the L1 region of all 19 high-risk genotypes, linear probe analysis was also used to detect and identify the papillomavirus genotypes (Bauer HM et al, "Detection of human papilloma viruses bypass reaction" US Patent No 569871).

Methods such as mass spectrometry are not feasible even at advanced tertiary care centers, and expensive scanner-based microarray detection is not possible in a clinical setting. Linear probe analysis is prone to amplicon contamination.

I. Definition of

"nucleotide" means a structural unit of DNA or RNA consisting of one base, one phosphate molecule and one sugar molecule (deoxyribose in DNA and ribose in RNA).

"oligonucleotide" means a short chain of nucleotides. Oligonucleotides are often used as probes for finding matching sequences of DNA or RNA and may be labeled with various labels, such as radioisotopes as well as fluorescent and chemiluminescent groups.

"primer" means a short strand of oligonucleotide complementary to a specific target sequence of DNA, which is used to initiate DNA synthesis.

"singleplex" means a PCR-based analysis using a single set of primers in each reaction that amplifies a single pathogen-specific DNA sequence.

By "multiplex" is meant a PCR-based assay that utilizes multiple primer sets in a single reaction, where each primer can amplify a single pathogen-specific DNA sequence.

The term "probe" refers to a DNA product (amplicon) resulting from PCR amplification of a target DNA.

The term "target" refers to a DNA sequence specific for a pathogen that is immobilized on an inert substrate such as nylon.

"hybridization" refers to the process of joining two complementary DNA strands to form a double-stranded molecule, and more specifically refers herein to the process between a "probe" and a "target" DNA sequence.

The term "detection system" as used herein refers to a method for visualizing the PCR amplified DNA product. Examples of suitable detection systems include systems that rely on color, radioactivity, fluorescence or chemiluminescence.

"pan-fungal" means a consensus gene sequence found in all pathogenic fungi-such as cryptococcus, candida, mucor, aspergillus, and rhizopus, etc. -and is used to identify any/all fungal species.

Disclosure of Invention

The present invention provides a series of chemically labeled pathogen-specific forward and reverse primers uniquely designed to specifically amplify target sequences from a pathogen in a multiplex polymerase chain reaction extended at a denaturation temperature of 95 ℃, an annealing temperature of 58-65 ℃, and 72 ℃. The invention also provides a series of target DNA sequences derived from pathogen-specific genes, which are immobilized on an inert carrier and specifically hybridize to PCR amplification products obtained using pathogen-specific forward and reverse PCR primers.

The present invention provides rapid assays for the simultaneous detection of pathogens causing infections of the eye and central nervous system for which immunological parameters do not show active infection but merely manifest as exposure to the pathogen, and for which classical microbiological assays such as bacterial and fungal cultures are neither sensitive enough to detect the pathogen nor rapid enough to identify the pathogen within 48 hours.

The present invention combines the high sensitivity of PCR analysis with the high specificity of discrimination methods for detecting hybridization by color detection by hybridization to macroarrays, the final result of which can be monitored by the naked eye. However, fluorescent labels such as Quantum Dots, Cy3, Cy5, and FITC can also be used, and the product can be observed by fluorescence microscopy.

The invention features multiplex assays for simultaneously detecting and identifying pathogens causing ocular and CNS infections, comprising:

i) clinical specimens from patients suspected of having an ocular or CNS infection (e.g., corneal shavings or conjunctival swabs or aqueous or vitreous humor or collected vitrectomy washes or cerebrospinal fluid aspirates or pus or epiretinal membranes collected from brain abscess material) were processed to isolate DNA by standard methods.

ii) amplifying specific DNA regions of genes specific for each pathogen by single-tube PCR technique using labelled amplification primers for each pathogen known to cause CNS and eye infections.

iii) detection and identification of pathogens by means of DNA hybridization, wherein the immobilized target sequence specific for each pathogen reacts with amplified DNA probes generated by a PCR reaction and hybridization is monitored by means of color development with a specific set of reagents.

In particular, the invention relates to the following:

1. primer sets for detection and identification of pathogens causing disorders:

group 1 is FP:5'cgcttggtttcggatgggag 3' (SEQ ID No.1)

RP：5′gcccccagagacttgttgtagg 3′(SEQ ID No.2)

Group 2 is FP:5'ggcaatcgtgtacgtcgtccg 3' (SEQ ID No.3)

RP：5′cgggggggtcttgcgttac 3′(SEQ ID No.4)

Group 3 is FP:5'caagctgacggacatttacaagg 3' (SEQ ID No.5)

RP：5′gtcccacacgcgaaacacg 3′(SEQ ID No.6)

Group 4 was FP:5'ttccggctcatggcgttaacc 3' (SEQ ID No.7)

RP：5′cgccctgcttttacgttacgc 3′(SEQ ID No.8)

Group 5 is FP:5'cggcgacgacgacgataaag 3' (SEQ ID No.9)

RP：5′caatctggtcgcgtaatcctctg 3′(SEQ ID No.10)

Group 6 was FP:5'gggcacgtcctcgcagaag 3' (SEQ ID No.11)

RP：5′ccaagatgcaggtgataggtgac 3′(SEQ ID No.12)

Group 7 is FP:5'ggtcttgccggagctggtattac 3' (SEQ ID No.13)

RP：5′tgcctccgtgaaagacaaagaca 3′(SEQ ID No.14)

Group 8 is FP:5'tccatttaacgttgcatcattttgtg 3' (SEQ ID No.15)

RP：5′acgttccggtagcgagttatctg 3′(SEQ ID No.16)

Group 9 was FP:5'cgccgccaacatgctctacc 3' (SEQ ID No.17)

RP：5′gttgcgggaggggatggata 3′(SEQ ID No.18)

Group 10 is FP:5'tgggctacacacgtgctacaatgg 3' (SEQ ID No.19)

RP：5′cggactacgatcggttttgtgaga 3′(SEQ ID No.20)

Group 11 was FP:5'ggcctaacacatgcaagtcgagc 3' (SEQ ID No.21)

RP：5′ggcagattcctaggcattactcacc 3′(SEQ ID No.22)

Group 12 is FP:5'acgtcaaatcatcatgcccccttat 3' (SEQ ID No.23)

RP：5′tgcagccctttgtaccgtccat 3′(SEQ ID No.24)

Group 13 is FP:5'gcggaacgtgggaccaatac 3' (SEQ ID No.25)

RP：5′cgacggggtgattttcttcttc 3′(SEQ ID No.26)

Group 14 was FP:5'aacttttttgactgccagacacactattg 3' (SEQ ID No.27)

RP：5’ggatgccaccccccaaaag 3′(SEQ ID No.28)

Group 15 was FP:5'tggttactcgcttggtgaatatgt 3' (SEQ ID No.29)

RP：5′gacgttttgccgactacctatcc 3′(SEQ ID No.30)

Group 16 was FP:5'cccctctgctggcgaaaagtg 3' (SEQ ID No.31)

RP：5′ggcgaccaatctgcgaatacac 3′(SEQ ID No.32)

Group 17 was FP:5 'aatcgtatctcgggttaatgttgc 3' (SEQ ID No.33)

RP：5’tcgaggaaaaccgtatgagaaac 3′(SEQ ID No.34)

Group 18 was FP:5'gctgggactgaggactgcgac 3' (SEQ ID No.35)

RP：5′ttcaagacgggcggcatataac 3′(SEQ ID No.36)

Group 19 is FP:5'tggcgaacgggtgagtaaca 3' (SEQ ID No.37)

RP：5′ccggtattagccccagtttcc 3′(SEQ ID No.38)

Group 20 was FP:5'cggcggcaagttcgacgac 3' (SEQ ID No.39)

RP：5′ccaccgagacgcccacacc 3′(SEQ ID No.40)

Group 21 is FP:5'ccaggtcggcggagaagc 3' (SEQ ID No.41)

RP：5′ccaccggcccgatgacc 3′(SEQ ID No.42)

Group 22 is FP:5'gccgccctgaccaccttc 3' (SEQ ID No.43)

RP：5′gcgggttgttcggcatcag 3′(SEQ ID No.44)

Wherein the primers are used alone or in combination.

2. The primer set according to item 1, which can be used for detecting and identifying a pathogen causing infectious endophthalmitis or keratitis or uveitis or retinitis or meningitis by amplifying a target gene of the pathogen by performing a multiplex polymerase chain reaction on a clinical specimen.

3. The primer set according to item 1, which has a uniform annealing temperature in the range of 58 ℃ to 65 ℃ and a length in the range of 17 to 29 bases.

4. A set of pathogen-specific DNA probe sequences as listed below, amplified from a standard or clinical sample by a singleplex or multiplex PCR reaction using the primers described in item 1:

1) probe DNA sequence

“cgcttggtttcggatgggaggcaactgtgctatccccatcacggtcatggagtacaccgaatgctcctacaacaagtctctgggggc”(SEQ ID No.45)

2) Probe DNA sequence

“ggcaatcgtgtacgtcgtccgcacatcacagtcgcggcagcgtcatcggcggtaacgcaagacccccccg”(SEQ ID No.46)

3) Probe DNA sequence

“caagctgacggacatttacaaggtccccctggacgggtacggccgcatgaacggccggggcgtgtttcgcgtgtgggac”(SEQ ID No.47)

4) Probe DNA sequence

“ttccggctcatggcgttaaccaggtagaaactgtgtgtacagttgcgttgtgcgtaacgtaaaagcagggcg”(SEQ ID No.48)

5) Probe DNA sequence

“cggcgacgacgacgataaagaatacaaagccgcagtgtcgtccagaggattacgcgaccagat tg”(SEQ ID No.49)

6) Probe DNA sequence

“gggcacgtcctcgcagaaggactccaggtacaccttgacgtactggtcacctatcacctgcatcttgg”(SEQ ID No.50)

7) Probe DNA sequence

“ggtcttgccggagctggtattaccttaaaactcactaccagtcatttctatccatctgtctttgtctttcacggaggca”(SEQ ID No.51)

8) Probe DNA sequence

“tccatttaacgttgcatcattttgtgttatcatagaactgcgtaaacactcggcaagtaatacagataactcgctaccggaacgt”(SEQ ID No.52)

9) Probe DNA sequence

“cgccgccaacatgctctaccctatacccgccaacgctaccaacgtgcccatatccatcccctcccgcaac”(SEQ ID No.53)

10) Probe DNA sequence

“tgggctacacacgtgctacaatggtcggtacagagggtcgccaaaccgcgaggtggagctaatctcacaaaaccgatcgtagtccg”(SEQ ID No.54)

11) Probe DNA sequence

“ggcctaacacatgcaagtcgagcggatgaaaggagcttgctcctggattcagcggcggacgggtgagtaatgcctaggaatctgcc”(SEQ ID No.55)

12) Probe DNA sequence

“acgtcaaatcatcatgcccccttatgacctgggctacacacgtgctacaatggacggtacaaagggctgca”(SEQID No.56)

13) Probe DNA sequence

“gcggaacgtgggaccaatacctgggttgggccggctgcttcgggcagcaactcccccgggttgaagaagaaaatcaccccgtcg”(SEQ ID No.57)

14) Probe DNA sequence

“aacttttttgactgccagacacactattgggctttgagacaacaggcccgtgccccttttggggggtggcatcc”(SEQ ID No.58)

15) Probe DNA sequence

“tggttactcgcttggtgaatatgttttataaatcctgtccaccccgtggataggtagtcggcaaaacgtc”(SEQ ID No.59)

16) Probe DNA sequence

“cccctctgctggcgaaaagtgaaattcatgagtatctgtgcaactttggtgtattcgcagattggtcgcc”(SEQ ID No.60)

17) Probe DNA sequence

“aatcgtatctcgggttaatgttgcatgatgctttatcaaatgacaagcttagatccgtttctcatacggttttcctcga”(SEQ ID No.61)

18) Probe DNA sequence

“gctgggactgaggactgcgacgtaagtcaaggatgctggcataatggttatatgccgcccgtcttgaa”(SEQ ID No.62)

19) Probe DNA sequence

“tggcgaacgggtgagtaacacgtgagtaacctgcccttgactttgggataacttcaggaaactggggctaataccgg”(SEQ ID No.63)

20) Probe DNA sequence

“cggcggcaagttcgacgacaacacctacaaggtgtccggcggcttgcacggtgtgggcgtctcggtgg”(SEQ ID No.64)

21) Probe DNA sequence

“ccaggtcggcggagaagccgaggcaggcgaggtccttcagttcgtcgcgggtcatcgggccg gtgg”(SEQ ID No.65)

22) Probe DNA sequence

“gccgccctgaccaccttcatcagcctggccggccgttacctggtgctgatgccgaacaacccgc”(SEQ ID No.66)。

5. A set of DNA target sequences listed below, having a uniform melting temperature in the range of 58.9 ℃ to 83 ℃, complementary to the amplification sequences used after immobilization on a solid substrate during hybridization to further detect and identify the particular pathogen under study:

1)5’gcaactgtgctatccccatcacggtcatggagtacaccgaatgct3’(SEQ ID No.67)

2)5’cacatcacagtcgcggcagcgtcatcggcg 3’(SEQ ID No.68)

3)5’tccccctggacgggtacggccgcatgaacggccgggg 3’(SEQ ID No.69)

4)5’aggtagaaactgtgtgtacagttgcgttgtg 3’(SEQ ID No.70)

5)5’aatacaaagccgcagtgtcgtc 3’(SEQ ID No.71)

6)5’gactccaggtacaccttgacgtactg 3’(SEQ ID No.72)

7)5’cttaaaactcactaccagtcatttctatccatc 3’(SEQ ID No.73)

8)5’ttatcatagaactgcgtaaacactcggcaagtaata 3’(SEQ ID No.74)

9)5’ctatacccgccaacgctaccaacgtgccca 3’(SEQ ID No.75)

10)5’tcggtacagagggtcgccaaaccgcgaggtggagctaa 3’(SEQ ID No.76)

11)5’ggatgaaaggagcttgctcctggattcagcggcggacg 3’(SEQ ID No.77)

12)5’gacctgggctacacacgtgctaca 3’(SEQ ID No.78)

13)5’ctgggttgggccggctgcttcgggcagcaactcccccgggtt 3’(SEQ ID No.79)

14)5’ggctttgagacaacaggcccgtgccc 3’(SEQ ID No.80)

15)5’tttataaatcctgtccaccccgt 3’(SEQ ID No.81)

16)5’aaattcatgagtatctgtgcaactttg 3’(SEQ ID No.82)

17)5’atgatgctttatcaaatgacaagcttagatcc 3’(SEQ ID No.83)

18)5’gtaagtcaaggatgctggcataatg 3’(SEQ ID No.84)

19)5’gcttcagcgccgtcagcgaggataac 3’(SEQ ID No.85)

20)5’aacacctacaaggtgtccggcggcttgcac 3’(SEQ ID No.86)

21)5’cgaggcaggcgaggtccttcagttcgtcgcg 3’(SEQ ID No.87)

22)5’atcagcctggccggccgttacctggtg 3’(SEQ ID No.88)。

6. a method for detecting and identifying pathogens causing conditions such as infectious endophthalmitis or keratitis or uveitis or retinitis or meningitis, wherein a specific gene of the pathogen is amplified by performing multiplex PCR (polymerase chain reaction) on a clinical sample using a unique set of forward and reverse primer sets as described in item 1, and in a further step, the amplified product (probe) is hybridized by known methods to complementary DNA sequences (targets) immobilized in multiplex form on a solid substrate, and the hybridized product is detected by known methods to further detect and identify the specific pathogen causing the condition in question.

7. Multiplex PCR assay using all or some of the primer sets described in item 1, where all primers can be used together in a single tube using uniform thermocycling conditions characterized by a denaturation step of 94 ℃ for 5 minutes followed by 40 cycles of 60 ℃ -64 ℃ for 45 seconds, 72 ℃ for 45 seconds and 94 ℃ for 45 seconds and 72 ℃ for 10 minutes of extension reaction.

8. The method as described in item 6, wherein the primer is labeled with a biotin group at the 5' end, resulting in detection by formation of a colored product.

9. The method as described in item 6, wherein the primer is labeled by fluorescence, for example, an organic fluorescent label such as fluorescein isothiocyanate or a Quantum Dots^TMOr Cy3 or Cy5, enabling detection by any fluorescence scanning device or microscopy method.

10. Use of a primer set as described in item 1, a probe as described in item 4, a target as described in item 5, wherein the assay is real-time PCR for detection of a pathogen.

11. Use of a primer set as described in item 1, a probe as described in item 4, a target as described in item 5, wherein the analysis is real-time PCR for quantifying pathogens in a clinical sample for monitoring prognosis or treatment of a disease.

12. Detection of amplification products as described in item 6 with a specific probe as described in item 4, in the form of macroarray or slot hybridization or linear probe analysis.

13. A macroarray as claimed in claim 12 consisting of targets as claimed in claim 5 immobilized on a solid phase comprising nitrocellulose, nylon, charged nylon, glass or polystyrene.

14. The detection method as described in item 12, wherein the pathogens to be detected are herpes simplex viruses 1 and 2, cytomegalovirus, varicella zoster virus, adenovirus, eubacteria, gram positive organisms, gram negative bacteria, fungi, tubercle bacillus, mycobacterium cheloniae, mycobacterium fortuitum, toxoplasma gondii, chlamydia trachomatis.

15. The detection method as described in item 6, which enables detection of individual pathogens from the group of possible pathogens described in item 14 causing diseases of the eye or nervous system with similar manifestations.

16. A multiplex PCR assay employing some selected or all of the primers as described in item 1, wherein any clinical symptoms caused by some or all of the organisms as described in item 14 are studied to detect any one individual pathogen or group of pathogens present in a clinical sample.

17. A method for simultaneously detecting all pathogens causing external eye infections, endophthalmitis or uveitis or retinitis or meningoencephalitis, comprising the steps of:

a) clinical samples were taken from patients with infection,

b) extracting DNA from a part or all of the clinical specimen obtained in step a),

c) performing multiplex PCR on the template DNA obtained in step b) using a primer set as described in item 1 labeled with biotin or a fluorescent tracer and standard PCR reagents,

d) denaturing the PCR product obtained in step c),

e) hybridizing the PCR product to a target as described in item 5 immobilized on a solid substrate,

f) detecting the DNA hybrids obtained in step e) on the solid substrate by enzymatic or fluorescent means.

18. A kit for the simultaneous detection of all pathogens causing external eye infections, endophthalmitis or uveitis or retinitis or meningoencephalitis, comprising:

a) the set of forward and reverse primers as described in item 1,

b) a substrate of the DNA sequence as described in item 5 immobilized on a suitable solid phase,

c) standard reagents required for amplification of DNA by polymerase chain reaction,

d) standard reagents required for hybridizing the PCR amplification product to a matrix of DNA sequences immobilized on a suitable solid phase,

e) standard reagents required for detection and identification of the final hybridization product.

Brief description of the drawings

FIG. 1 shows a schematic view of a: 6% agarose gel electrophoresis images showing single and multiplex PCR amplification products of the adenovirus hexon gene and the Chlamydia trachomatis genome. Wherein, the following steps: NC-negative control; 1-positive control-adenovirus; 2-positive control-chlamydia trachomatis; 3-positive control-multiplex PCR (adenovirus and chlamydia trachomatis); MW-100bp DNA ladder.

FIG. 2: a4% agarose gel electrophoresis image showing the amplification product of Herpes Simplex Virus (HSV) glycoprotein D, DNA polymerase, UL-44 region. Way: NC-negative control; 1-positive control glycoprotein D region; 2-positive control DNA polymerase region; 3-positive control UL-44 region; MW-100bp ladder-like band.

FIG. 3: 6% agarose gel electrophoresis images showing multiplex PCR amplification products of external eye infection. Way: NC-negative control; 1-positive control (HSV); 2-positive control (chlamydia trachomatis); 3-positive control (adenovirus); 4-positive control (all three genomes); MW-HinfI digest of DNA.

FIG. 4: and (3) a macro-array photo on a nylon membrane hybridized with the multiplex PCR amplicon for identification of external eye infection of genome for specific recognition of HSV, Chlamydia trachomatis and adenovirus. DNA array (left to right): showing the template spotted with the DNA probe on the nylon membrane. HSV-herpes simplex virus, showing that the spot is marked as HGD ═ HSV glycoprotein D, HDP ═ HSVDNA polymerase, HUL ═ UL 44 gene; 1^stComp, complementary strand probe of HGD, CT-chlamydia trachomatis, AV-adenovirus. NC-negative control, HSV-membrane hybridized with the amplicon of tube 1, CT-membrane hybridized with the amplicon of tube 2, AV-membrane hybridized with the amplicon of tube 3.

FIG. 5: macroarray photographs on nylon membranes hybridized with multiplex PCR amplicons for detection of uveitis and other suspected mycobacterial infections specifically recognizing genomes of toxoplasma gondii, mycobacterium tuberculosis, mycobacterium fortuitum and mycobacterium cheloni. From left to right, the first is a template showing how the target is spotted on the membrane. MBT-tuberculosis, MBF-Mycobacterium fortuitum, MBC-Mycobacterium chelonii, TG-Toxoplasma gondii. Second, NC hybridized to a negative control tube labeled NC, nylon membranes hybridized to amplicons obtained from tubes 1, 2, 3 and 4 were labeled MBT, MBF, MBC and TG, respectively.

FIG. 6A photograph of a macroarray on a nylon membrane hybridized with amplicons of a multiplex PCR for the identification of viral retinitis that specifically recognizes the genomes of HSV, CMV and VZV. From left to right, show how the probe dots the template on each nylon membrane. HSV-herpes simplex virus, shown as HGD ═ HSV glycoprotein D, HDP ═ HSV DNA polymerase, HUL ═ UL 44 gene, 1^stThe complementary strand probe of HGD; CMV-cytomegalovirus, shown as CMT ═ morphotropic gene II, CGO ═ cytomegalovirus glycoprotein O, CUL ═ UL83 gene, 5^thA complementary strand probe for comp ═ CMT; VZV-varicella zoster virus, shown as VO ═ varicella zoster ORF29 gene, VDP ═ varicella zoster DNA polymerase; NC: a membrane that hybridizes to the contents of a tube labeled NC; HSV-nylon membrane hybridized with amplicon obtained from tube No. 1; CMV-Nylon Membrane hybridized to the amplicon obtained from tube No.2 and VZV-Nylon Membrane hybridized to the contents of tube No. 3.

FIG. 7A photograph of macroarrays on nylon membranes hybridized with amplicons of multiplex PCR for genomic identification of infectious endophthalmitis, in particular eubacteria, gram-positive bacteria, gram-negative bacteria, propionibacterium acnes and fungi. NC ═ negative control, GN showed ERR ═ 16s ribosomes of eubacterium group IRNA genes, ERW ═ 16s ribosomal RNA gene of eubacteria group II, GN31 ═ gyr B gene of gram-negative bacteria, GN67 ═ aconitate hydratase gene of gram-negative bacteria, GN87 ═ ribonuclease gene of gram-negative bacteria, GP ═ 16s ribosomal RNA gene of gram-positive bacteria, PA ═ propionibacterium acnes 16s ribosomal RNA gene, PF ═ fungi 28s ribosomal RNA gene. The left corner of the top end is a probe spotting template. NC is a nylon membrane hybridized to a negative control tube. GN, GP, PA and PF are nylon membranes hybridized to the amplicons of tubes 1, 2, 3 and 4, respectively.

Detailed Description

The method is suitable for designing the unique PCR primer of the pathogen DNA multiplex PCR of the clinical sample of the eye infection patient.

The following genes from various pathogens known to cause eye infections were selected based on known information in the literature.

1. Herpes simplex virus 1&2 glycoprotein D

2. Herpes simplex virus 1&2UL 44 gene

3. Herpes simplex virus 1&2DNA polymerase gene

4. Cytomegalovirus glycoprotein O gene

5. Cytomegalovirus morphotropic transformation gene

6. Cytomegalovirus UL88 gene

7. Varicella zoster ORF29

8. Varicella zoster DNA polymerase gene

9. Adenovirus Hexon (Hexon) gene

10. Eubacterium 16s ribosomal RNA gene I

11. Eubacterium 16s ribosomal RNA gene region II

Gram-positive bacteria-specific part of the 12.16s ribosomal RNA gene

13. Mycobacterium tuberculosis (MPB 64) gene

14. Mycobacterium fortuitum (Mycobacterium fortuitum)16s-23s RNA gene

15. Mycobacterium cheloni (Mycobacterium chelonei)16s-23s RNA gene

16. Toxoplasma gondii (Toxoplasma gondii) B1 gene

17. Chlamydia trachomatis (Chlamydia trachomatis) polymorphic protein (polymorphic protein) II

Fungal specific portions of 18.28s ribosomal RNA genes

Propionibacterium acnes (Propionibacterium acnes) specific portion of the 19.16s-23s ribosomal RNA gene

Gram-negative bacterium-specific part of the gyr B gene

21. Aconitic acid hydratase gene of gram-negative bacterium

22. Gram-negative ribonuclease 1 gene

To further increase the reliability of detecting certain organisms (e.g., herpes simplex 1 and 2, cytomegalovirus, varicella zoster, and gram negative bacteria), more than one gene from each organism was selected for amplification. In the case of herpes simplex 1 and 2 and cytomegalovirus, three different genes were selected for each organism, while for varicella zoster, two genes were selected.

The DNA polymerase gene of herpes virus is a gene that confers PCR sensitivity and is used in many studies. In the first study, the 179bp product was amplified using thermocycling conditions of denaturation at 95 ℃ for 45 seconds, annealing at 64 ℃ for 45 seconds, and extension at 72 ℃ for 45 seconds (Madhavan HN et al, Detection of Human Simple Virus (HSV) using a Polymerase Chain Reaction (PCR) in a clinical samples company of PCR with standard laboratory methods for the Detection of HSV, J.Clin.Virol.14: 145-151 (1999)). In yet another study, the 469 and 391bp regions of the same gene were amplified using different primer sets and thermocycling conditions (denaturation at 95 ℃ for 45 seconds, annealing at 60 ℃ for 45 seconds and extension at 72 ℃ for 45 seconds) (Madhavan HN et al, photoprotective and Genotypic methods for the detection of peptides in simplex virospopes J. Virol. methods, 108: 97-102. (2003)). In summary, different thermal conditions are used in the detection of different viruses, as in the case of PCR for the identification of HSV, CMV and VZV of eye infections (Priya K et al, Association of viruses in aqueous humor of tissues with serogenetic science: a polymerase chain reaction based study, Ocular Immunology and fluorescence 9: 1-9 (2003)). In this study, the reaction conditions and primer concentrations were different for different viruses. It is therefore evident that it is difficult to design primers and specific target sequences for a range of known pathogens to enable a single-tube multiplex PCR reaction that allows for rapid detection and identification of one or more pathogens in a given clinical sample.

Therefore, it is considered necessary to explore the possibility of designing suitable PCR primers and target DNA sequences complementary to PCR amplification products using known bioinformatics methods.

To achieve this, the present invention first determines the following conditions for multiplex PCR reactions that are preferentially used for detection of HSV, CMV and VZV, i.e., denaturation at 95 ℃ followed by annealing at 58 ℃ -65 ℃ and then extension at 72 ℃. The optimal hybridization temperature of the PCR amplification product thus obtained with each pathogen-specific target DNA sequence immobilized on a solid phase substrate was determined at 48 ℃ to 55 ℃. It is therefore considered necessary to design the set of target DNA sequences of each pathogen under investigation so that the specific PCR amplification product hybridizes to its complementary target DNA sequence at a uniform temperature without non-specific binding of the DNA sequences.

The most difficult part in designing primers for multiplex PCR reactions is to design all primers to have the same melting temperature, thereby making it possible to amplify all genes at the same thermal cycling temperature.

These primer sets for amplification were selected from the gene sequences mentioned above so that all primers had annealing temperatures in the range of 58-65 ℃ so that all 22 genes could be amplified in the same tube by PCR, which always represented all strains or genotypes of the particular pathogen under study.

Amplicons of different sizes may interfere with the efficiency of multiplex amplification of the PCR method. The second criterion for selecting primers is therefore determined by the uniform size of the amplicons in the 66-90 nucleotide range. Regions comprising a length of 66-90bp (including primer sequences) were selected for all the genes mentioned in the above section.

To keep the melting temperature uniform, the primer length was varied between 17-29 base pairs.

In addition, in multiplex reactions, looping or cross-hybridization in the primers due to the presence of complementary regions may interfere with the PCR amplification itself. To avoid all this complication, all primers are carefully designed to eliminate looping or primer cross-hybridization between themselves. Care is also taken to avoid any non-specific (cross-) amplification of the primer sets, i.e. the primers of one organism/gene should not react with the genes of any other organism/gene in the reaction mixture.

All primers were designed such that they generally matched all nucleotide bases of the pathogen gene. However, if there is a mismatch in certain strains or species, as in the case of primers designed to amplify gram-positive, gram-negative bacteria and fungi, the mismatch is limited to a maximum of two nucleotides in the middle of the primer. The 3' end of each primer ensures that there is always a perfect match in all strains of the species diagnosed.

In addition to the described criteria, general criteria for selecting primer sequences in the prior art are cited here, in Molecular cloning: this is described in detail in A Laboratory manual, Vol 2, Sambrook.J., Russell DW (Eds) Cold spring Harbor Laboratory Press NY (2001). These criteria are that the 3' end is G or C, avoiding tandem GC repeats and generally any primer does not terminate with a T, etc.

After design, primers were used individually and in multiplex to test sensitivity and specificity, using the standard DNA sequences (genes) of all pathogens listed. In the identification of some organisms or virus particles, different sets of primers are selected using the same criteria when the sensitivity is below the level reported in the prior art, e.g. Madhavan HN, et al, Detection of Human Simple Virus (HSV) using a polymerase reaction (PCR) in a clinical samples company of PCR with a standard analysis method for the Detection of HSV, J.Clin.virol.14: 145-151 (1999); medical research, 117: 71-75 (2003); the enzyme antibody of polymerase reaction (PCR) and DNA probe hybridization to the interaction of the interacting bacteria in the intracellular fluids of tissues with the endogenous antibody of Infection, 41: 221-226 (2000); use of polymerase Chain reaction in fundamental endophthalmitis 108: 326-330(2001). Even though some primers anneal at 58 ℃, it is experimentally ensured that all primers produce good amplification at 60 ℃.

In the present embodiment, the following were selected through careful evaluationUnique primerAnd for detection and identification of pathogens. These sequences are unique and unknown in the prior art.

1. Amplification of the herpes simplex virus 1 and 2 glycoprotein D genes by primer set 1 comprising SEQ ID Nos. 1 and 2 (FP: 5'cgcttggtttcggatgggag 3' (SEQ ID No.1) and RP: 5 'gcccccagagacttgttgtagg 3' (SEQ ID No.2))

2. Amplification of the herpes simplex virus 1 and 2UL 44 genes by primer set 2 comprising SEQ ID Nos. 3 and 4 (FP: 5'ggcaatcgtgtacgtcgtccg 3' (SEQ ID No.3) and RP: 5 'cgggggggtcttgcgttac 3' (SEQ ID No.4))

3. Amplification of the herpes simplex virus 1 and 2DNA polymerase genes by primer set 3 comprising SEQ ID Nos. 5 and 6 (FP: 5'caagctgacggacatttacaagg 3' (SEQ ID No.5) and RP: 5 'gtcccacacgcgaaacacg 3' (SEQ ID No.6))

4. Amplification of the Cytomegalovirus glycoprotein O Gene by primer set 4 comprising SEQ ID Nos. 7 and 8 (FP: 5'ttccggctcatggcgttaacc 3' (SEQ ID No.7) and RP: 5 'cgccctgcttttacgttacgc 3' (SEQ ID No.8))

5. Amplification of Cytomegalovirus morphotropic genes by primer set 5 comprising SEQ ID Nos. 9 and 10 (FP: 5'cggcgacgacgacgataaag 3' (SEQ ID No.9) and RP: 5 'caatctggtcgcgtaatcctctg 3' (SEQ ID No.10))

6. Amplification of the Cytomegalovirus UL88 Gene by primer set 6 comprising SEQ ID Nos. 11 and 12 (FP: 5'gggcacgtcctcgcagaag 3' (SEQ ID No.11) and RP: 5 'ccaagatgcaggtgataggtgac 3' (SEQ ID No.12))

7. Varicella zoster ORF29 was amplified by primer set 7 comprising SEQ ID Nos. 13 and 14 (FP: 5'ggtcttgccggagctggtattac 3' (SEQ ID No.13) and RP: 5 'tgcctccgtgaaagacaaagaca 3' (SEQ ID No.14))

8. Varicella zoster DNA polymerase gene was amplified by primer set 8 comprising SEQ ID Nos. 15 and 16 (FP: 5'tccatttaacgttgcatcattttgtg 3' (SEQ ID No.15) and RP: 5 'acgttccggtagcgagttatctg 3' (SEQ ID No.16))

9. Amplification of adenovirus hexon genes by primer set 9 comprising SEQ ID Nos. 17 and 18 (FP: 5'cgccgccaacatgctctacc 3' (SEQ ID No.17) and RP: 5 'gttgcgggaggggatggata 3' (SEQ ID No.18))

10. Amplification of eubacterium 16s ribosomal RNA gene region I by primer set 10 comprising SEQ ID Nos. 19 and 20 (FP: 5'tgggctacacacgtgctacaatgg 3' (SEQ ID No.19) and RP: 5 'cggactacgatcggttttgtgaga 3' (SEQ ID No.20))

11. Amplification of eubacterium 16s ribosomal RNA gene region II by primer set 11 comprising SEQ ID Nos. 21 and 22 (FP: 5'ggcctaacacatgcaagtcgagc 3' (SEQ ID No.21) and RP: 5 'ggcagattcctaggcattactcacc 3' (SEQ ID No.22))

12. Amplification of gram-positive bacterium-specific portion of the 16s ribosomal RNA gene by primer set 12 comprising SEQ ID Nos. 23 and 24 (FP: 5'acgtcaaatcatcatgcccccttat 3' (SEQ ID No.23) and RP: 5 'tgcagccctttgtaccgtccat 3' (SEQ ID No.24))

13. Amplification of the Mycobacterium tuberculosis MPB 64 Gene by primer set 13 comprising SEQ ID Nos. 25 and 26 (FP: 5'gcggaacgtgggaccaatac 3' (SEQ ID No.25) and RP: 5 'cgacggggtgattttcttcttc 3' (SEQ ID No.26))

14. Amplification of the Mycobacterium fortuitum 16s-23s RNA gene by primer set 14 comprising SEQ ID Nos. 27 and 28 (FP: 5'aacttttttgactgccagacacactattg 3' (SEQ ID No.27) and RP: 5 'ggatgccaccccccaaaag 3' (SEQ ID No.28))

15. Amplification of the Mycobacterium chelonii 16s-23s RNA gene by primer set 15 comprising SEQ ID Nos. 29 and 30 (FP: 5'tggttactcgcttggtgaatatgt 3' (SEQ ID No.29) and RP: 5 'gacgttttgccgactacctatcc 3' (SEQ ID No.30))

16. Amplification of the Toxoplasma gondii B1 gene by primer set 16 comprising SEQ ID Nos. 31 and 32 (FP: 5'cccctctgctggcgaaaagtg 3' (SEQ ID No.31) and RP: 5 'ggcgaccaatctgcgaatacac 3' (SEQ ID No.32))

17. Amplification of Chlamydia trachomatis polymorphic protein II by primer set 17 comprising SEQ ID Nos. 33 and 34 (FP: 5 'aatcgtatctcgggttaatgttgc 3' (SEQ ID No.33) and RP: 5 'tcgaggaaaaccgtatgagaaac 3' (SEQ ID No.34))

18. Amplification of the fungus-specific part of the 28s ribosomal RNA gene by means of a primer set 18 comprising SEQ ID Nos. 35 and 36 (FP: 5' gctgggactgaggactgcgac 3' (SEQ ID No.35) and RP: 5' ttcaagacgggcggcatataac 3(SEQ ID No.36))

19. By including SEQ ID Nos. 37 and 38: (FP：5'tggcgaacgggtgagtaaca 3' (SEQ ID No.37) and RP: 5 'ccggtattagccccagtttcc 3' (SEQ ID No.38)) of the primer set 19 amplified the 16s-23s ribosomal RNA gene for Propionibacterium acnesSex part

20. Amplification of the gram-negative bacterium-specific portion of the gyr B gene by primer set 20 comprising SEQ ID Nos. 39 and 40 (FP: 5'cggcggcaagttcgacgac 3' (SEQ ID No.39) and RP: 5 'ccaccgagacgcccacacc 3' (SEQ ID No.40))

21. Amplification of Aconite hydratase gene of gram-negative bacterium by primer set 21 comprising SEQ ID Nos. 41 and 42 (FP: 5'ccaggtcggcggagaagc 3' (SEQ ID No.41) and RP: 5 'ccaccggcccgatgacc 3' (SEQ ID No.42))

22. Amplification of gram-negative ribonuclease 1 Gene by primer set 22 comprising SEQ ID Nos. 43 and 44 (FP: 5'gccgccctgaccaccttc 3' (SEQ ID No.43) and RP: 5 'gcgggttgttcggcatcag 3' (SEQ ID No.44))

In another embodiment of the invention, having SEQ ID Nos. 45-66Probe sequenceObtained by a computer program for designing primers recognizing specific gene segments unique to said pathogen. The length of the probe sequence varies between 66-90 nucleotides. The probe itself does not form a hairpin loop therein. They do not share homology with any other amplicons. The probe may be amplified from either strand of the pathogen's DNA.

The probe sequences are detailed below:

1. probe DNA sequence "cgcttggtttcggatgggaggcaactgtgctatccccatcacggtcatggagtacaccgaatgctcctacaacaagtctctgggggc" (SEQ ID No.45) of the herpes simplex virus 1 and 2 glycoprotein D genes (amplified by primer set 1 comprising FP:5'cgcttggtttcggatgggag 3' (SEQ ID No.1) and RP: 5 'gcccccagagacttgttgtagg 3' (SEQ ID No.2))

2. Probe DNA sequence "ggcaatcgtgtacgtcgtccgcacatcacagtcgcggcagcgtcatcggcggtaacgcaagaccccc ccg" (SEQ ID No.46) of herpes simplex virus 1 and 2UL 44 genes (amplified by primer set 2 comprising FP:5'ggcaatcgtgtacgtcgtccg 3' (SEQ ID No.3) and RP: 5 'cgggggggtcttgcgttac 3' (SEQ ID No.4))

3. Probe DNA sequence "caagctgacggacatttacaaggtccccctggacgggtacggccgcatgaacggccggggcgtgtttcgcgtgtgggac" (SEQ ID No.47) of herpes simplex virus 1 and 2DNA polymerase genes (amplified by primer set 3 comprising FP:5'caagctgacggacatttacaagg 3' (SEQ ID No.5) and RP: 5 'gtcccacacgcgaaacacg 3' (SEQ ID No.6))

4. Probe DNA sequence "ttccggctcatggcgttaaccaggtagaaactgtgtgtacagttgcgttgtgcgtaacgtaaaagcagg gcg" (SEQ ID No.48) of the Cytomegalovirus glycoprotein O gene (amplified by primer set 4 comprising FP:5'ttccggctcatggcgttaacc 3' (SEQ ID No.7) and RP: 5 'cgccctgcttttacgttacgc 3' (SEQ ID No.8))

5. Probe DNA sequence "cggcgacgacgacgataaagaatacaaagccgcagtgtcgtccagaggattacgcgaccagattg" (SEQ ID No.49) of the cytomegalovirus morphological transformation region II gene (amplified by primer set 5 comprising FP:5'cggcgacgacgacgataaag 3' (SEQ ID No.9) and RP: 5 'caatctggtcgcgtaatcctctg 3' (SEQ ID No.10))

6. Probe DNA sequence "gggcacgtcctcgcagaaggactccaggtacaccttgacgtactggtcacctatcacctgcatcttgg" (SEQ ID No.5o) of Cytomegalovirus UL88 gene (amplified by primer set 6 comprising FP:5'gggcacgtcctcgcagaag 3' (SEQ ID No.11) and RP: 5 'ccaagatgcaggtgataggtgac 3' (SEQ ID No.12))

7. Probe DNA sequence "ggtcttgccggagctggtattaccttaaaactcactaccagtcatttctatccatctgtctttgtctttcacggaggca" (SEQ ID No.51) of varicella zoster ORF29 (amplified by primer set 7 comprising FP:5'ggtcttgccggagctggtattac 3' (SEQ ID No.13) and RP: 5 'tgcctccgtgaaagacaaagaca 3' (SEQ ID No.14))

8. Probe DNA sequence "tccatttaacgttgcatcattttgtgttatcatagaactgcgtaaacactcggcaagtaatacagataactcgctaccggaacgt" (SEQ ID No.52) of the varicella zoster DNA polymerase gene (amplified by primer set 8 comprising FP:5'tccatttaacgttgcatcattttgtg 3' (SEQ ID No.15) and RP: 5 'acgttccggtagcgagttatctg 3' (SEQ ID No.16))

9. Probe DNA sequence "cgccgccaacatgctctaccctatacccgccaacgctaccaacgtgcccatatccatcccctcccgca ac" (SEQ ID No.53) for the adenovirus hexon gene (amplified by primer set 9 comprising FP:5'cgccgccaacatgctctacc 3' (SEQ ID No.17) and RP: 5 'gttgcgggaggggatggata 3' (SEQ ID No.18))

10. Probe DNA sequence "tgggctacacacgtgctacaatggtcggtacagagggtcgccaaaccgcgaggtggagctaatctcacaaaaccgatcgtagtccg" (SEQ ID No.54) of eubacterium 16s ribosomal RNA gene region I (amplified by primer set 10 comprising FP:5'tgggctacacacgtgctacaatgg 3' (SEQ ID No.19) and RP: 5 'cggactacgatcggttttgtgaga 3' (SEQ ID No.20))

11. Probe DNA sequence "ggcctaacacatgcaagtcgagcggatgaaaggagcttgctcctggattcagcggcggacgggtgagtaatgcctaggaatctgcc" (SEQ ID No.55) of eubacterium 16s ribosomal RNA gene region II (amplified by primer set 11 comprising FP:5'ggcctaacacatgcaagtcgagc 3' (SEQ ID No.21) and RP: 5 'ggcagattcctaggcattactcacc 3' (SEQ ID No.22))

Probe DNA sequence "acgtcaaatcatcatgcccccttatgacctgggctacacacgtgctacaat ggacggtacaaagggctgca" (SEQ ID No.56) of the gram-positive bacterium-specific part of the 12.16s ribosomal RNA gene (amplified by primer set 12 comprising FP:5'acgtcaaatcatcatgcccccttat 3' (SEQ ID No.23) and RP: 5 'tgcagccctttgtaccgtccat 3' (SEQ ID No.24))

13. Probe DNA sequence "gcggaacgtgggaccaatacctgggttgggccggctgcttcgggcagcaactcccccgggttgaagaagaaaatcaccccgtcg" (SEQ ID No.57) of Mycobacterium tuberculosis MPB 64 gene (amplified by primer set 13 comprising FP:5'gcggaacgtgggaccaatac 3' (SEQ ID No.25) and RP: 5 'cgacggggtgattttcttcttc 3' (SEQ ID No.26))

14. Probe DNA sequence "aacttttttgactgccagacacactattgggctttgagacaacaggcccgtgccccttttggggggtggc atcc" (SEQ ID No.58) of Mycobacterium fortuitum 16s-23s RNA gene (amplified by primer set 14 comprising FP:5'aacttttttgactgccagacacactattg 3' (SEQ ID No.27) and RP: 5 'ggatgccaccccccaaaag 3' (SEQ ID No.28))

15. Probe DNA sequence "tggttactcgcttggtgaatatgttttataaatcctgtccaccccgtggataggtagtcggcaaaacgtc" (SEQ ID No.59) of Mycobacterium chelonii 16s-23s RNA gene (amplified by primer set 15 comprising FP:5'tggttactcgcttggtgaatatgt 3' (SEQ ID No.29) and RP: 5 'gacgttttgccgactacctatcc 3' (SEQ ID No.30))

16. Probe DNA sequence "cccctctgctggcgaaaagtgaaattcatgagtatctgtgcaactttggtgtattcgcagattggtcgcc" (SEQ ID No.60) of the Toxoplasma gondii B1 gene (amplified by primer set 16 comprising FP:5'cccctctgctggcgaaaagtg 3' (SEQ ID No.31) and RP: 5 'ggcgaccaatctgcgaatacac 3' (SEQ ID No.32))

17. Probe DNA sequence "aatcgtatctcgggttaatgttgcatgatgctttatcaaatgacaagcttagatccgtttctcatacggttttcctcga" (SEQ ID No.61) of Chlamydia trachomatis polymorphic protein II (amplified by primer set 17 comprising FP:5 'aatcgtatctcgggttaatgttgc 3' (SEQ ID No.33) and RP: 5 'tcgaggaaaaccgtatgagaaac 3' (SEQ ID No.34))

Probe DNA sequence "gctgggactgaggactgcgacgtaagtcaaggatgctggcataatggttatatgccgcccgtcttgaa" (SEQ ID No.62) of the fungal-specific part of the 18.28s ribosomal RNA gene (amplified by primer set 18 comprising FP:5' gctgggactgaggactgcgac 3' (SEQ ID No.35) and RP: 5' ttcaagacgggcggcatataac 3(SEQ ID No.36))

19.16s-23s ribosomal RNA Gene Probe DNA sequence "tggcgaacgggtgagtaacacgtgagtaacctgcccttgactttgggataacttcaggaaactggggctaataccgg" (SEQ ID No.63) for the Propionibacterium acnes-specific portion (by includingFP：5'tggcgaacgggtgagtaaca 3' (SEQ ID No.37) and RP: primer set 19 amplification of 5 'ccggtattagccccagtttcc 3' (SEQ ID No.38)

Probe DNA sequence "cggcggcaagttcgacgacaacacctacaaggtgtccggcggcttgcacggtgtgggcgtctc ggtgg" (SEQ ID No.64) of gram-negative bacterium-specific part of the Gyr B gene (amplified by primer set 20 comprising FP:5'cggcggcaagttcgacgac 3' (SEQ ID No.39) and RP: 5 'ccaccgagacgcccacacc 3' (SEQ ID No.40))

21. Probe DNA sequence "ccaggtcggcggagaagccgaggcaggcgaggtccttcagttcgtcgcgggtcatcgggccggtg g" (SEQ ID No.65) of Aconite hydratase gene of gram-negative bacterium (amplified by primer set 21 comprising FP:5'ccaggtcggcggagaagc 3' (SEQ ID No.41) and RP: 5 'ccaccggcccgatgacc 3' (SEQ ID No.42))

22. Probe DNA sequence "gccgccctgaccaccttcatcagcctggccggccgttacctggtgctgatgccgaacaacccgc" (SEQ ID No.66) of gram-negative ribonuclease 1 gene (amplified by primer set 22 comprising FP:5'gccgccctgaccaccttc 3' (SEQ ID No.43) and RP: 5 'gcgggttgttcggcatcag 3' (SEQ ID No.44))

It seems to repeat one pass.

In another embodiment of the invention, having SEQ ID Nos. 67-88Target sequenceGenerated from the probe sequence using a computer program. These targets are used to immobilize on inert materials such as nylon and are cross-linked or chemically immobilized using UV radiation. The targets were selected according to the following criteria:

1. all target sequences are pathogen-specific and do not overlap with any other sequences of other pathogens.

2. All target sequences are in the range of 23-28 bases in length.

3. All targets have a uniform melting temperature in the range of 58.9 ℃ to 88 ℃.

4. The target sequence falls within the amplicon region and does not contain forward or reverse primer sequences, so that the labeled probes (having SEQ ID nos. 45-66) do not bind non-specifically to these targets.

5. All targets are designed in such a way that they generally match all nucleotide bases. However, if there is a mismatch in some probes, the mismatch is limited to a maximum of two nucleotides in the middle of the probe.

The target sequences are described in detail below:

1.5 'gcaactgtgctatccccatcacggtcatggagtacaccgaatgct 3' (SEQ ID No.67), HSV glycoprotein D target amplified by primer set 1

2.5 'cacatcacagtcgcggcagcgtcatcggcg 3' (SEQ ID No.68), HSV UL 44 target amplified by primer set 2

3.5 'tccccctggacgggtacggccgcatgaacggccgggg 3' (SEQ ID No.69), HSV polymerase target amplified by primer set 3

4.5 'aggtagaaactgtgtgtacagttgcgttgtg 3' (SEQ ID No.70), CMV glycoprotein O target amplified by primer set 4

5.5 'aatacaaagccgcagtgtcgtc 3' (SEQ ID No.71), Cytomegalovirus morphotropically transforming Gene II targets amplified by primer set 5

6.5 'gactccaggtacaccttgacgtactg 3' (SEQ ID No.72), targets of the cytomegalovirus UL88 Gene amplified by primer set 6

7.5 'cttaaaactcactaccagtcatttctatccatc 3' (SEQ ID No.73), the target of varicella zoster Virus ORF29 gene amplified by primer set 7

8.5 'ttatcatagaactgcgtaaacactcggcaagtaata 3' (SEQ ID No.74), a target of varicella zoster Virus DNA polymerase gene amplified by primer set 8

9.5 'ctatacccgccaacgctaccaacgtgccca 3' (SEQ ID No.75), adenovirus hexon gene targets amplified by primer set 9

10.5 'tcggtacagagggtcgccaaaccgcgaggtggagctaa 3' (SEQ ID No.76), target of region I of the eubacterium 16s ribosomal RNA gene amplified by the primer set 10

11.5 'ggatgaaaggagcttgctcctggattcagcggcggacg 3' (SEQ ID No.77), eubacterium 16s ribosomal RNA gene region II target amplified by primer set 11

12.5 'gacctgggctacacacgtgctaca 3' (SEQ ID No.78), target of the 16s ribosomal RNA gene of gram-positive organisms amplified by primer set 12

13.5 'ctgggttgggccggctgcttcgggcagcaactcccccgggtt 3' (SEQ ID No.79), target of Mycobacterium tuberculosis MPB 64 gene amplified by primer set 13

14.5 'ggctttgagacaacaggcccgtgccc 3' (SEQ ID No.80), a target of the 16s-23s RNA gene of Mycobacterium fortuitum amplified by the primer set 14

15.5 'tttataaatcctgtccaccccgt 3' (SEQ ID No.81), the target of Mycobacterium chelonii 16s-23s RNA gene amplified by primer set 15

16.5 'aaattcatgagtatctgtgcaactttg 3' (SEQ ID No.82), target of the Toxoplasma gondii B1 gene amplified by the primer set 16

17.5 'atgatgctttatcaaatgacaagcttagatcc 3' (SEQ ID No.83), target of Chlamydia trachomatis polymorphic protein II amplified by primer set 17

18.5 'gtaagtcaaggatgctggcataatg 3' (SEQ ID No.84), all fungal 28s ribosomal RNA gene targets amplified by primer set 18

19.5 'gcttcagcgccgtcagcgaggataac 3' (SEQ ID No.85), target of the 16s ribosomal RNA gene of Propionibacterium acnes amplified by primer set 19

20.5 'aacacctacaaggtgtccggcggcttgcac 3' (SEQ ID No.86), the target of the gyrase gene of gram-negative organisms amplified by the primer set 20

21.5 'cgaggcaggcgaggtccttcagttcgtcgcg 3' (SEQ ID No.87), targets of aconitate hydratase genes of gram-negative organisms amplified by the primer set 21

22.5 'atcagcctggccggccgttacctggtg 3' (SEQ ID No.88), a target of a ribonuclease gene of a gram-negative organism amplified by the primer set 2, 2

The oligonucleotides reported above for immobilization on inert matrices were confirmed using products generated from standard DNA as well as clinical samples (by sequence analysis). These sequences are unique and unknown or have not been mentioned for use in multiplex or singleplex PCR.

In yet another embodiment of the present invention, a multiplex PCR assay using all or some of the above primer sets is provided, wherein all primers can be used together in a single tube using uniform thermal cycling conditions, including a denaturation step at 94 ℃ for 5 minutes, followed by 40 cycles of 60 ℃ -64 ℃ for 45 seconds, 72 ℃ for 45 seconds and 94 ℃ for 45 seconds, and an extension reaction at 72 ℃ for 10 minutes.

In a further embodiment, a primer set labeled with biotin at the 5' end allows detection of colored products.

In yet another embodiment, the primer is labeled with a fluorescent label (e.g., an organic fluorescent label such as fluorescein isothiocyanate FITC or such as Quantum Dots)^TMOr inorganic fluorescent nanoparticles of Cy3 or Cy 5) to allow detection by any fluorescent scanning device or microscope.

In another embodiment, the invention provides the use of the primer and probe set wherein the assay is real-time PCR for the detection of pathogens.

In yet another embodiment, the invention provides the use of the primer and probe set wherein the assay is real-time PCR for the quantitative determination of pathogens in clinical samples to monitor prognosis or treatment of disease.

In yet another embodiment, the invention provides the use of said primer set, wherein the detection of the amplification products may be in the form of a macroarray or slot hybridization or a linear probe assay.

In a further embodiment, the present invention provides a macroarray consisting of said probes immobilized on a solid phase comprising nitrocellulose, nylon, charged nylon, glass or polystyrene.

In another embodiment, the invention provides a method for detecting and identifying pathogens that cause conditions such as infectious endophthalmitis or keratitis or uveitis or retinitis or meningitis, wherein the pathogens detected are herpes simplex virus 1 and 2, cytomegalovirus, varicella zoster virus, adenovirus, eubacteria, gram positive organisms, gram negative bacteria, fungi, tubercle bacillus, mycobacterium chelonii, mycobacterium fortuitum, toxoplasma gondii, chlamydia trachomatis.

In yet another embodiment, the invention provides a method for detecting an individual pathogen among a group of possible pathogens causing diseases of the eye or the central system with similar symptoms.

In yet another embodiment, the invention provides any multiplex PCR assay using selected ones or all of the foregoing primers, wherein any clinical symptoms caused by some or all of the organisms are studied for the detection of any one individual pathogen or group of pathogens present in a clinical sample.

In a further embodiment, the present invention provides a method for simultaneously detecting all pathogens causing external eye infections, endophthalmitis or uveitis or retinitis or meningoencephalitis, comprising the steps of:

a) taking a clinical sample from a patient suffering from said infection,

b) extracting DNA from a part or all of the sample obtained in step a),

c) performing multiplex PCR on the DNA obtained in step b) using a primer set as defined in claim 1 labeled with biotin or a fluorescent tracer and standard PCR reagents,

d) denaturing the PCR product obtained in step c),

e) hybridizing the PCR product obtained in step d) with a target immobilized on a solid substrate,

f) detecting the DNA hybrids on the solid substrate obtained in step e) by enzymatic or fluorescent methods.

In another embodiment, the invention provides a kit for simultaneously detecting all pathogens that cause external eye infection, endophthalmitis or uveitis or retinitis or meningoencephalitis, comprising:

a) the set of forward and reverse primers as described above,

b) a DNA target matrix as described above immobilized on a suitable solid support,

c) standard reagents required for amplification of DNA by polymerase chain reaction,

d) standard reagents required for hybridizing the PCR amplification product to the immobilized substrate of the DNA probe,

e) standard reagents required for detection and identification of the final hybridization product of a pathogen of a particular etiology are tested.

In a further embodiment, the invention provides a method for simultaneously detecting all pathogens that cause external eye infection, endophthalmitis or uveitis or retinitis or meningoencephalitis, comprising:

a) taking a clinical sample from a patient suffering from said infection,

b) extracting DNA from a part or all of the sample obtained in step a),

c) performing multiplex PCR on the DNA obtained in step b) using a primer set labeled with biotin or a fluorescent tracer as described above and standard PCR reagents,

d) denaturing the PCR product obtained in step c),

e) hybridizing the PCR product obtained in step d) with a target as described before immobilized on a solid substrate,

f) detecting the DNA hybrids on the solid substrate obtained in step e) by enzymatic or fluorescent methods.

Examples

The following examples are given by way of illustration of the present invention and therefore should not be construed to limit the scope of the present invention.

Example 1:

multiplex PCR was performed using primer sets 9 and 17 capable of amplifying the adenovirus hexon gene and the C.trachomatis polymorphic protein II gene, respectively. The PCR mix contained 10-20pmol of each forward and reverse primer, 200. mu.M of each d-ATP, d-UTP, d-CTP and d-GTP, 2 units of Taq polymerase in 10mM Tris-HCl (pH9.0), 1.5mM MgCl₂5mM KCl, 0.01% gelatin, 1mM EDTA and 1 unit UDP glycosylase (to prevent amplicon contamination). Cycling conditions were incubation at 37 ℃ for 30 minutes to completely digest any amplicon contaminants, 2 minutes at 50 ℃, 5 minutes at 94 ℃ denaturation step followed by 40 cycles of 60 ℃ for 45 seconds, 72 ℃ for 45 seconds and 94 ℃ for 45 seconds and 10 minutes at 72 ℃ extension reaction. The products were analyzed by 6% agarose gel. As seen in fig. 1, both genes were amplified. Standard DNA of 1pg adenovirus and 10fg Chlamydia DNA was used for amplification.

Example 2:

multiplex PCR was performed using primer sets 1, 2 and 3 capable of amplifying glycoprotein D, UL 44 and DNA polymerase genes, respectively. The PCR mix contained 10pmol of each forward and reverse primer, 200. mu.M of each d-ATP, d-UTP, d-CTP and d-GTP, 2 units of Taq polymerase in 10mM Tris-HCl (pH 7.5), 1.5mM MgCl₂5mM KCl, 0.01% gelatin, 1mM EDTA and 1 unit UDP glycosylase (to prevent amplicon contamination). Cycling conditions were incubation at 37 ℃ for 30 minutes to completely digest any amplicon contaminants, 2 minutes at 50 ℃, 5 minutes at 94 ℃ denaturation step followed by 40 cycles of 60 ℃ for 45 seconds, 72 ℃ for 45 seconds and 94 ℃ for 45 seconds and 10 minutes at 72 ℃ extension reaction. The products were analyzed by 6% agarose gel. As seen in fig. 2, all three genes were amplified.

Example 3

Multiplex PCR was performed using primer sets 1, 2, 3, 9 and 17 capable of amplifying HSV glycoprotein D gene, UL 44 gene and DNA polymerase gene, adenovirus hexon gene and chlamydia trachomatis polymorphic protein II gene, respectively. The PCR mix contained 10pmol of each forward and reverse primer, 200. mu.M of each d-ATP, d-UTP, d-CTP and d-GTP, 2 units of Taq polymerase in 10mM Tris-HCl (pH9.0), 1.5mM MgCl₂5mM KCl, 0.01% gelatin, 1mM EDTA and 1 unit UDP glycosylase (to prevent amplicon contamination). Cycling conditions were incubation at 37 ℃ for 30 minutes to completely digest any amplicon contaminants, 2 minutes at 50 ℃, 5 minutes at 94 ℃ denaturation step followed by 40 cycles of 60 ℃ for 45 seconds, 72 ℃ for 45 seconds and 94 ℃ for 45 seconds and 10 minutes at 72 ℃ extension reaction. 5 tubes the PCR mixture described above was incubated with the following DNA preparation, where tube NC had no DNA added, tube 1 contained 1pg of HSV DNA, tube 2 contained 4fg of Chlamydia trachomatis, tube 3 contained 10pg of adenovirus DNA, and tube 4 contained all three of the above amounts of DNA. The products were analyzed by 6% agarose gel. As seen in fig. 3, all genes were amplified.

Nylon membrane spots with 100pmol of the target with SEQ ID Nos. 67, 68, 69, 75 and 83 in 0.26N NaOH (FIG. 4). The membrane was then blocked using 2X SSPE containing 0.1% SDS and 1% BSA for 1 hour at 37 ℃. Amplicons were heated to 95 ℃ for 10 min, mixed in 2X SSPE containing 0.1% SDS and hybridized at 52 ℃ for 2 h. After hybridization, the membranes were washed 5 times for 3 minutes each in 1X SSPE containing 0.1% SDS. The membrane was incubated with streptavidin-peroxidase conjugate in 0.1M Tris-HCl (pH 7.4) containing 1% BSA, 150mM NaCl and 0.3% tween-20. After 30 minutes at 37 ℃, the membranes were washed 3 minutes each with the same buffer and 5 times. For color development, the membrane was incubated with 0.5 mg/ml phosphate buffered saline diaminobenzidine for 10 minutes at 37 ℃. The appearance of a brown stain indicates the presence of a particular pathogen.

Example 4:

multiplex PCR was performed using primer sets 13, 14, 15 and 16 capable of amplifying the MPB 64 gene of Mycobacterium tuberculosis, the 16s-23s RNA gene of Mycobacterium fortuitum, the 16s-23s RNA gene of Mycobacterium chelonii and the B1 gene of Toxoplasma gondii, respectively. The PCR mix contained 10pmol of each forward and reverse primer, 200. mu.M of each d-ATP, d-UTP, d-CTP and d-GTP, 2 units of Taq polymerase in 10mM Tris-HCl (pH9.0), 1.5mM MgCl₂5mM KCl, 0.01% gelatin, 1mM EDTA and 1 unit UDP glycosylase (to prevent amplicon contamination). Cycling conditions were incubation at 37 ℃ for 30 minutes to completely digest any amplicon contaminants, 2 minutes at 50 ℃, 5 minutes at 94 ℃ denaturation step followed by 40 cycles of 60 ℃ for 45 seconds, 72 ℃ for 45 seconds and 94 ℃ for 45 seconds and 10 minutes at 72 ℃ extension reaction. 5 tubes PCR mix as described above was incubated with the following DNA preparation, where tube NC did not contain any DNA, tube 1 contained 1fg of Mycobacterium tuberculosis DNA, tube 2 contained 100fg of Mycobacterium fortuitum DNA, tube 3 contained 100fg of Mycobacterium tortoise DNA, and tube 4 contained 1fg of Toxoplasma gondii DNA. FIG. 5 shows five nylon membranes, each spotted with 100pmol of the target of SEQ ID Nos. 79, 80, 81 and 82 in 0.26N NaOH. The membrane was then blocked using 2X SSPE containing 0.1% SDS and 1% BSA for 1 hour at 37 ℃. The amplicons were heated to 95 ℃ for 10 minutes, mixed in 1ml 2 XSSPE containing 0.1% SDS and hybridized at 52 ℃ for 2 hours. After hybridization, the membranes were washed 5 times for 3 minutes each in 1X SSPE containing 0.1% SDS. The membrane was incubated with streptavidin-peroxidase conjugate in 0.1M Tris-HCl (pH 7.4) containing 1% BSA, 150mM NaCl and 0.3% tween-20. After 30 minutes at 37 ℃, the membranes were washed 3 minutes each with the same buffer and 5 times. For color development, the membrane was incubated with 0.5 mg/ml phosphate buffered saline diaminobenzidine for 10 minutes at 37 ℃. The appearance of a brown stain indicates the presence of a particular pathogen.

Example 5

Using glycoprotein D gene, UL 44 gene and DNA polymerase gene capable of amplifying HSV, glycoprotein O gene of CMV, morphological transformation andmultiplex PCR was performed using primer sets 1, 2, 3, 4, 5, 6, 7 and 8 for the UL88 gene and the ORF29 gene and DNA polymerase gene of VZV. The PCR mix contained 10pmol of each forward and reverse primer, 200. mu.M of each d-ATP, d-UTP, d-CTP and d-GTP, 2 units of Taq polymerase in 10mM Tris-HCl (pH9.0), 1.5mM MgCl₂5mM KCl, 0.01% gelatin, 1mM EDTA and 1 unit UDP glycosylase (to prevent amplicon contamination). Cycling conditions were incubation at 37 ℃ for 30 minutes to completely digest any amplicon contaminants, a denaturation step at 50 ℃ for 2 minutes, and 94 ℃ for 5 minutes, followed by 40 cycles of 60 ℃ for 45 seconds, 72 ℃ for 45 seconds, 94 ℃ for 45 seconds, and an extension reaction at 72 ℃ for 10 minutes.

4 tubes the PCR mix described above was incubated with the following DNA preparation, where tube NC did not contain any DNA, tube 1 contained 1pg of HSV DNA, tube 2 contained 10pg of CMV DNA, and tube 3 contained 1pg of VZV DNA. FIG. 6 shows four pieces of nylon membrane, each with 100pmol of the target of SEQ ID Nos. 67, 68, 69, 70, 71, 72, 73 and 74 in 0.26N NaOH. The membrane was then blocked using 2X SSPE containing 0.1% SDS and 1% BSA for 1 hour at 37 ℃. Amplicons were heated to 95 ℃ for 10 min, mixed in 2X SSPE containing 0.1% SDS and hybridized at 52 ℃ for 2 h. After hybridization, the membranes were washed 5 times for 3 minutes each in 1X SSPE containing 0.1% SDS. The membrane was incubated with streptavidin-peroxidase conjugate in 0.1M Tris-HCl (pH 7.4) containing 1% BSA, 150mM NaCl and 0.3% tween-20. After 30 minutes at 37 ℃, the membranes were washed 3 minutes each with the same buffer and 5 times. For color development, the membrane was incubated with 0.5 mg/ml phosphate buffered saline diaminobenzidine for 10 minutes at 37 ℃. The appearance of a brown stain indicates the presence of a particular pathogen.

Example 6

16s ribosomal RNA genes I and II capable of amplifying eubacterial genome, 16s ribosomal RNA gene of gram-positive bacterium, 28sRNA gene of all fungi, 16s ribosomal RNA gene of Propionibacterium acnes, gyrB gene of gram-negative bacterium, aconitic acid, respectively, were usedThe primer sets 10, 11, 12, 18, 19, 20, 21 and 22 for the hydratase gene and ribonuclease gene were subjected to multiplex PCR. The PCR mix contained 10-20pmol of each forward and reverse primer, 200. mu.M of each d-ATP, d-UTP, d-CTP and d-GTP, 2 units of Taq polymerase in 50mM Tris-HCl (pH 7.5), 5mM MgCl₂5mM KCl, 0.01% bovine serum albumin, 1mM EDTA and 1 unit of UDP glycosylase (to prevent amplicon contamination). Cycling conditions were incubation at 37 ℃ for 30 minutes to completely digest any amplicon contaminants, a 5 minute denaturation step at 94 ℃ followed by 40 cycles of 60 ℃ for 45 seconds, 72 ℃ for 45 seconds and 94 ℃ for 45 seconds and an extension reaction at 72 ℃ for 10 minutes. Tube 5 PCR mixtures as described above were incubated with the following DNA preparation, where tube NC did not contain any DNA, tube 1 contained 5fg of e.coli (e.coli) DNA, tube 2 contained 10fg of s.aureus (s.aureus) DNA, tube 3 contained 10fg of p.acnes (p.acnes) DNA, and tube 4 contained 10fg of candida albicans (c.albicans) DNA. FIG. 7 shows five nylon membranes, each spotted with 100pmol of the target of SEQ ID Nos. 76, 77, 78, 84, 85, 86, 87 and 88 in 0.26N NaOH. The membrane was then blocked using 2X SSPE containing 0.1% SDS and 1% BSA for 1 hour at 37 ℃. Amplicons were heated to 95 ℃ for 10 min, mixed in 2X SSPE containing 0.1% SDS and hybridized at 52 ℃ for 2 h. After hybridization, the membranes were washed 5 times for 3 minutes each in 1X SSPE containing 0.1% SDS. The membrane was incubated with streptavidin-peroxidase conjugate in 0.1M Tris-HCl (pH 7.4) containing 1% BSA, 150mM NaCl and 0.3% tween-20. After 30 minutes at 37 ℃, the membranes were washed 3 minutes each with the same buffer and 5 times. For color development, the membrane was incubated with 0.5 mg/ml phosphate buffered saline diaminobenzidine for 10 minutes at 37 ℃. The appearance of a brown stain indicates the presence of a particular pathogen.

Example 7

Vitreous humor was collected from necropsies of 11 AIDS patients with pre-mortem uveitis/retinitis, samples were tested on multiplex PCR, and amplicons were subsequently identified on a macroarray. DNA was extracted from 100. mu.l of each vitreous sample using the QIAGEN DNA purification kit. The DNA was reconstituted in 50. mu.l of elution buffer. Multiplex PCR is carried out using a primer set 1-22 capable of amplifying all 22 genes, i.e., herpes simplex virus 1&2 glycoprotein D, herpes simplex virus 1&2UL 44 gene, herpes simplex virus 1&2DNA polymerase gene, cytomegalovirus glycoprotein O gene, cytomegalovirus morphology-converting gene, cytomegalovirus UL88 gene, varicella zoster ORF29, varicella zoster DNA polymerase gene, adenovirus hexon gene, eubacterium 16s ribosomal RNA gene I, eubacterium 16s ribosomal RNA gene region II, gram-positive bacterium-specific part of 16s ribosomal RNA gene, Mycobacterium tuberculosis MPB 64 gene, Mycobacterium fortuitum 16s-23s RNA gene, Mycobacterium chelonii 16s-23s RNA gene, Toxoplasma B1 gene, Chlamydia trachomatis polymorphic protein II, Mycobacterium tuberculosis MPB 64 gene, Mycobacterium fortuitum DNA, DNA polymerase gene, DNA polymerase chain reaction, and the like, A fungus-specific part of 28s ribosomal RNA gene, a Propionibacterium acnes-specific part of 16s-23s ribosomal RNA gene, a gram-negative bacterium-specific part of gyr B gene, a gram-negative bacterium aconitate hydratase gene, and a gram-negative bacterium ribonuclease I gene.

The PCR mix contained 10-20pmol of each forward and reverse primer, 200. mu.M of each d-ATP, d-UTP, d-CTP and d-GTP, 2 units of Taq polymerase in 10mM Tris-HCl (pH9.0), 1.5mM MgCl₂5mM KCl, 0.01% gelatin, 1mM EDTA and 1 unit UDP glycosylase (to prevent amplicon contamination) and 10. mu.l DNA extracted from the sample. Cycling conditions were incubation at 37 ℃ for 30 minutes to completely digest any amplicon contaminants, treatment at 50 ℃ for 2 minutes, and a denaturation step at 94 ℃ for 5 minutes followed by 40 cycles of 60 ℃ for 45 seconds, 72 ℃ for 45 seconds, and 94 ℃ for 45 seconds followed by an extension reaction at 72 ℃ for 10 minutes.

As described above, PCR was performed using 22 sets of primers including SEQ ID NO.1-44 at a concentration of 10-20 pmol/50. mu.l of the reaction mixture. The PCR products of all samples were hybridized on nylon membranes spotted with probes of SEQ ID Nos. 45-66. Nylon membrane dots with 0.26N NaOH 100pmol with SEQ ID No.67-88 target. The membrane was then blocked using 2X SSPE containing 0.1% SDS and 1% BSA for 1 hour at 37 ℃. Amplicons were heated to 95 ℃ for 10 min, mixed in 2X SSPE containing 0.1% SDS and hybridized at 52 ℃ for 2 h. After hybridization, the membranes were washed 5 times for 3 minutes each in 1X SSPE containing 0.1% SDS. The membrane was incubated with streptavidin peroxidase linker in 0.1M Tris-HCl (pH 7.4) containing 1% BSA, 150mM NaCl and 0.3% tween-20. After 30 minutes at 37 ℃, the membranes were washed 3 minutes each with the same buffer and 5 times. For color development, the membrane was incubated with 0.5 mg/ml phosphate buffered saline diaminobenzidine for 10 minutes at 37 ℃. The appearance of a brown stain indicates the presence of a particular pathogen

The results obtained are summarized in table 1. All 11 samples were identified as HSV retinitis and uveitis of tubercle bacillus, with another toxoplasma gondii in the 10 sample vitreous. Multiplex PCR and DNA macroarrays accurately identified all samples.

TABLE 1: results of simultaneous pathogen detection and identification using multiplex PCR and hybridization on macroarrays on 11 necropsy vitreous humor samples obtained from AIDS patients

Sample identification number	Positive for organism
		A/39/06	HSV, tubercle bacillus, toxoplasma

A/40/06	HSV, tubercle bacillus, toxoplasma
		A/05/06	HSV, tubercle bacillus, toxoplasma
A/12/06	HSV, tubercle bacillus, toxoplasma
		A/36/06	HSV, tubercle bacillus, toxoplasma
A/38/05	HSV, tubercle bacillus, toxoplasma
		A/14/06	HSV, tubercle bacillus, toxoplasma
A/42/05	HSV, tubercle bacillus, toxoplasma
		A/43/05	HSV, tubercle bacillus, toxoplasma
A/49/05	HSV，TB

Example 8:

six CSF samples from AIDS patient necropsies were tested on multiplex PCR followed by macroarray analysis. The cause of death was identified as a central nervous system infection. DNA was extracted from 200. mu.l of the sample using a commercially available QIAGEN DNA extraction kit. The DNA was reconstituted in 50. mu.l of elution buffer. Multiplex PCR is carried out using a primer set 1-22 capable of amplifying all 22 genes, i.e., herpes simplex virus 1&2 glycoprotein D, herpes simplex virus 1&2UL 44 gene, herpes simplex virus 1&2DNA polymerase gene, cytomegalovirus glycoprotein O gene, cytomegalovirus morphology-converting gene, cytomegalovirus UL88 gene, varicella zoster ORF29, varicella zoster DNA polymerase gene, adenovirus hexon gene, eubacterium 16s ribosomal RNA gene I, eubacterium 16s ribosomal RNA gene region II, gram-positive bacterium-specific part of 16s ribosomal RNA gene, Mycobacterium tuberculosis MPB 64 gene, Mycobacterium fortuitum 16s-23s RNA gene, Mycobacterium cheloniae 16s-23sRNA gene, Toxoplasma B1 gene, Chlamydia trachomatis polymorphic protein II, fungal-specific part of 28s ribosomal RNA gene, DNA sequence, A Propionibacterium acnes specific part of a 16s-23s ribosomal RNA gene, a gram-negative bacterium specific part of a gyr B gene, a gram-negative bacterium aconitate hydratase gene, and a gram-negative bacterium ribonuclease I gene.

The PCR mix contained 10-20pmol of each forward and reverse primer, 200. mu.M of each d-ATP, d-UTP, d-CTP and d-GTP, 2 units of Taq polymerase in 10mM Tris-HCl (pH9.0), 1.5mM MgCl₂5mM KCl, 0.01% gelatin, 1mM EDTA and 1 unit UDP glycosylase (to prevent amplicon contamination) and 10. mu.l DNA extracted from the sample. Cycling conditions were incubation at 37 ℃ for 30 minutes to completely digest any amplicon contaminants, treatment at 50 ℃ for 2 minutes, and a denaturation step at 94 ℃ for 5 minutes followed by 40 cycles of 60 ℃ for 45 seconds, 72 ℃ for 45 seconds, and 94 ℃ for 45 seconds followed by an extension reaction at 72 ℃ for 10 minutes. As described above, PCR was performed using 22 sets of primers including SEQ ID NO.1-44 at a concentration of 10-20 pmol/50. mu.l of the reaction mixture. The PCR products of all samples were hybridized on a nylon membrane spotted with the targets of SEQ ID Nos. 67-88 in 100pmol of 0.26N NaOH. The membrane was then blocked using 2X SSPE containing 0.1% SDS and 1% BSA for 1 hour at 37 ℃. Amplicons were heated to 95 ℃ for 10 min, mixed in 2X SSPE containing 0.1% SDS and hybridized at 52 ℃ for 2 h. Hetero compoundAfter cross-linking, the membranes were washed 5 times for 3 minutes each in 1X SSPE containing 0.1% SDS. The membrane was incubated with streptavidin-peroxidase conjugate in 0.1M Tris-HCl (pH 7.4) containing 1% BSA, 150mM NaCl and 0.3% tween-20. After 30 minutes at 37 ℃, the membranes were washed 3 minutes each with the same buffer and 5 times. For color development, the membrane was incubated with 0.5 mg/ml phosphate buffered saline diaminobenzidine for 10 minutes at 37 ℃. The appearance of a brown stain indicates the presence of a particular pathogen

TABLE 2: results of simultaneous pathogen detection and identification using multiplex PCR and hybridization on macroarrays on 6 necropsy CSF samples obtained from AIDS patients

Sample diagnosis	Number of tests	Number of positive
			HSV encephalitis	4	4
CMV encephalitis	2	2
			VZV encephalitis	2	2
Toxoplasmosis encephalitis	3	3
			Tubercular meningitis	3	3

Example 9:

a series (19) of eye samples (aqueous humor or vitreous humor) were obtained from various clinical diagnostic cases. DNA was extracted from approximately 50-100. mu.l of the sample using a commercially available DNA extraction kit and the DNA was reconstituted in 50. mu.l of water. Mu.l was used for multiplex PCR containing 10-20pmol of each primer set 1-22 (including SEQ ID Nos. 1-44). The PCR reagent composition and thermal cycling conditions were the same as described in examples 6 and 7 above. As described in the examples above, the amplicons hybridize to the targets having SEQ ID Nos. 67-88. The results are summarized below, which demonstrate the clinical utility of the primer sets and probes.

TABLE 3: results of simultaneous detection and identification of pathogens using multiplex PCR and hybridization on macroarrays on aqueous and vitreous humor samples from patients

Sample number	Clinical diagnosis	Results
			1	Viral retinitis	CMV
2	Viral retinitis and uveitis	Mycobacterium tuberculosis, Mycobacterium cheloni and VZV
			3	Infectious endophthalmitis	Eubacteria and gram-positive bacteria
4	Viral retinitis	HSV
			5	Infectious endophthalmitis	Eubacteria and gram-positive bacteria
6	Infectious endophthalmitis	Eubacteria and gram-positive bacteria
			7	Infectious endophthalmitis	Eubacteria and gram-positive bacteria
8	Infectious endophthalmitis	Eubacteria and gram-positive bacteria
			9	Infectious endophthalmitis	Eubacteria and gram-positive bacteria
10	Infectious endophthalmitis	Thin and thinBacteria and gram-positive bacteria
			11	Infectious endophthalmitis and uveitis	Fungal infections
12	Infectious endophthalmitis and uveitis	Negative of
			13	Infectious endophthalmitis	Propionibacterium acnes
14	Infectious endophthalmitis	Eubacteria and gram-positive bacteria
			15	Infectious endophthalmitis	Eubacteria and gram-positive bacteria
16	Infectious endophthalmitis and uveitis	Eubacteria and tubercle bacillus
			17	Infectious endophthalmitis	Eubacteria and gram-positive bacteria

18	Infectious endophthalmitis	Negative of
			19	Infectious endophthalmitis	Eubacteria and gram-positive bacteria

Advantages of the invention：

1. A highly efficient and time saving kit.

2. The identification of specific pathogens at the early stage of infection will help physicians select the appropriate treatment regimen for the spread and cure of the disease.

3. The identification of multiple infections from the same sample would also facilitate treatment with highly effective therapies using drug combinations.

Claims (27)

1. A primer set combination for detecting and identifying a pathogen in a sample that causes a condition selected from the group consisting of infectious endophthalmitis, keratitis, uveitis, retinitis, meningitis and encephalitis, wherein said combination consists of the following primer sets:

group 1 is FP:5'cgcttggtttcggatgggag 3' (SEQ ID No.1)

RP:5'gcccccagagacttgttgtagg 3'(SEQ ID No.2)

Group 2 is FP:5'ggcaatcgtgtacgtcgtccg 3' (SEQ ID No.3)

RP:5'cgggggggtcttgcgttac 3'(SEQ ID No.4)

Group 3 is FP:5'caagctgacggacatttacaagg 3' (SEQ ID No.5)

RP:5'gtcccacacgcgaaacacg 3'(SEQ ID No.6)

Group 4 are FP:5'ttccggctcatggcgttaacc 3' (SEQ ID No.7)

RP:5'cgccctgcttttacgttacgc 3'(SEQ ID No.8)

Group 5 is FP:5'cggcgacgacgacgataaag 3' (SEQ ID No.9)

RP:5'caatctggtcgcgtaatcctctg 3'(SEQ ID No.10)

Group 6 are FP:5'gggcacgtcctcgcagaag 3' (SEQ ID No.11)

RP:5'ccaagatgcaggtgataggtgac 3'(SEQ ID No.12)

Group 7 are FP:5'ggtcttgccggagctggtattac 3' (SEQ ID No.13)

RP:5'tgcctccgtgaaagacaaagaca 3'(SEQ ID No.14)

Group 8 are FP:5'tccatttaacgttgcatcattttgtg 3' (SEQ ID No.15)

RP:5'acgttccggtagcgagttatctg 3'(SEQ ID No.16)

Group 9 is FP:5'cgccgccaacatgctctacc 3' (SEQ ID No.17)

RP:5'gttgcgggaggggatggata 3'(SEQ ID No.18)

Group 10 is FP:5'tgggctacacacgtgctacaatgg 3' (SEQ ID No.19)

RP:5'cggactacgatcggttttgtgaga 3'(SEQ ID No.20)

Group 11 is FP:5'ggcctaacacatgcaagtcgagc 3' (SEQ ID No.21)

RP:5'ggcagattcctaggcattactcacc 3'(SEQ ID No.22)

Group 12 are FP:5'acgtcaaatcatcatgcccccttat 3' (SEQ ID No.23)

RP:5'tgcagccctttgtaccgtccat 3'(SEQ ID No.24)

Group 13 is FP:5'gcggaacgtgggaccaatac 3' (SEQ ID No.25)

RP:5'cgacggggtgattttcttcttc 3'(SEQ ID No.26)

Group 14 is FP:5'aacttttttgactgccagacacactattg 3' (SEQ ID No.27)

RP:5’ggatgccaccccccaaaag 3'(SEQ ID No.28)

Group 15 are FP:5'tggttactcgcttggtgaatatgt 3' (SEQ ID No.29)

RP:5'gacgttttgccgactacctatcc 3'(SEQ ID No.30)

Group 16 is FP:5'cccctctgctggcgaaaagtg 3' (SEQ ID No.31)

RP:5'ggcgaccaatctgcgaatacac 3'(SEQ ID No.32)

Group 17 is FP:5 'aatcgtatctcgggttaatgttgc 3' (SEQ ID No.33)

RP:5’tcgaggaaaaccgtatgagaaac 3'(SEQ ID No.34)

Group 18 is FP:5'gctgggactgaggactgcgac 3' (SEQ ID No.35)

RP:5'ttcaagacgggcggcatataac 3'(SEQ ID No.36)

Group 19 is FP:5'tggcgaacgggtgagtaaca 3' (SEQ ID No.37)

RP:5'ccggtattagccccagtttcc 3'(SEQ ID No.38)

Group 20 is FP:5'cggcggcaagttcgacgac 3' (SEQ ID No.39)

RP:5'ccaccgagacgcccacacc 3'(SEQ ID No.40)

Group 21 is FP:5'ccaggtcggcggagaagc 3' (SEQ ID No.41)

RP:5'ccaccggcccgatgacc 3'(SEQ ID No.42)

Group 22 is FP:5'gccgccctgaccaccttc 3' (SEQ ID No.43)

RP:5'gcgggttgttcggcatcag 3'(SEQ ID No.44)。

2. The primer set combination of claim 1, wherein the pathogen is selected from the group consisting of herpes simplex virus 1 and 2, cytomegalovirus, varicella zoster virus, adenovirus, eubacteria, gram negative bacteria, fungi, toxoplasma gondii and chlamydia trachomatis.

3. The primer set combination of claim 1, wherein the pathogen is selected from gram-positive bacteria.

4. The primer set combination of claim 1, wherein the pathogen is selected from the group consisting of mycobacterium tuberculosis, mycobacterium cheloni, and mycobacterium fortuitum.

5. The primer set combination according to claim 1, wherein the primer set combination consisting of set 1 of SEQ ID No.1 and SEQ ID No.2, set 2 of SEQ ID No.3 and SEQ ID No.4, set 3 of SEQ ID No.5 and SEQ ID No.6, set 4 of SEQ ID No.7 and SEQ ID No.8, set 5 of SEQ ID No.9 and SEQ ID No.10, set 6 of SEQ ID No.11 and SEQ ID No.12, set 7 of SEQ ID No.13 and SEQ ID No.14, and set 8 of SEQ ID No.15 and SEQ ID No.16 detects viral retinitis in a sample; or

Wherein the primer set combination consisting of set 1 of SEQ ID No.1 and SEQ ID No.2, set 2 of SEQ ID No.3 and SEQ ID No.4, set 3 of SEQ ID No.5 and SEQ ID No.6, set 9 of SEQ ID No.17 and SEQ ID No.18, and set 17 of SEQ ID No.33 and SEQ ID No.34 detects keratoconjunctivitis in a sample; or

Wherein the combination of primer sets consisting of set 13 of SEQ ID No.25 and SEQ ID No.26, set 14 of SEQ ID No.27 and SEQ ID No.28, set 15 of SEQ ID No.29 and SEQ ID No.30 and set 16 of SEQ ID No.31 and SEQ ID No.32 detects uveitis in the sample; or

Wherein a primer set combination consisting of set 10 of SEQ ID No.19 and SEQ ID No.20, set 11 of SEQ ID No.21 and SEQ ID No.22, set 12 of SEQ ID No.23 and SEQ ID No.24, set 18 of SEQ ID No.35 and SEQ ID No.36, set 19 of SEQ ID No.37 and SEQ ID No.38, set 20 of SEQ ID No.39 and SEQ ID No.40, set 21 of SEQ ID No.41 and SEQ ID No.42, and set 22 of SEQ ID No.43 and SEQ ID No.44 detects infective endophthalmitis in the sample; or

Wherein a primer set combination consisting of set 1 of SEQ ID No.1 and SEQ ID No.2, set 2 of SEQ ID No.3 and SEQ ID No.4, set 3 of SEQ ID No.5 and SEQ ID No.6, set 4 of SEQ ID No.7 and SEQ ID No.8, set 5 of SEQ ID No.9 and SEQ ID No.10, set 6 of SEQ ID No.11 and SEQ ID No.12, set 7 of SEQ ID No.13 and SEQ ID No.14, set 8 of SEQ ID No.15 and SEQ ID No.16, set 13 of SEQ ID No.25 and SEQ ID No.26, set 16 of SEQ ID No.31 and SEQ ID No.32, and set 18 of SEQ ID No.35 and SEQ ID No.36 detects meningeal encephalitis in a sample; or

Wherein the combination of primer sets consisting of set 10 of SEQ ID No.19 and SEQ ID No.20, set 11 of SEQ ID No.21 and SEQ ID No.22, set 12 of SEQ ID No.23 and SEQ ID No.24, set 18 of SEQ ID No.35 and SEQ ID No.36, set 20 of SEQ ID No.39 and SEQ ID No.40, set 21 of SEQ ID No.41 and SEQ ID No.42 and set 22 of SEQ ID No.43 and SEQ ID No.44 detects gram positive and/or gram negative bacteria in the sample; or

Wherein a primer set combination consisting of set 10 of SEQ ID No.19 and SEQ ID No.20, set 11 of SEQ ID No.21 and SEQ ID No.22, set 12 of SEQ ID No.23 and SEQ ID No.24, set 13 of SEQ ID No.25 and SEQ ID No.26, set 18 of SEQ ID No.35 and SEQ ID No.36, set 20 of SEQ ID No.39 and SEQ ID No.40, set 21 of SEQ ID No.41 and SEQ ID No.42 and set 22 of SEQ ID No.43 and SEQ ID No.44 distinguishes pathogens of acute and chronic meningitis in a sample, wherein said chronic meningitis is caused by tubercle bacilli and various fungi.

6. The primer set combination according to claim 1, wherein the primer sets 1 to 22(SEQ ID Nos. 1 to 44) detect target nucleic acids of herpes simplex viruses 1 and 2, cytomegalovirus, varicella zoster, adenovirus, eubacteria, gram positive bacteria, gram negative bacteria, fungi, Mycobacterium tuberculosis, Mycobacterium cheloniae, Mycobacterium fortuitum, Toxoplasma gondii and Chlamydia trachomatis in a specimen.

7. The primer set combination of claim 6, wherein the sample is selected from the group consisting of aqueous humor, vitreous humor, vitrectomy wash, corneal scrapings, conjunctival swabs, and cerebrospinal fluid.

8. The primer set combination of claim 1, wherein the primers are labeled with a biotin group at the 5' end, resulting in detection by formation of a colored product; or the primer is labeled with a fluorescent label selected from the group consisting of an organic fluorescent label and an inorganic fluorescent label to enable detection by any equipment or microscopy method.

9. The primer set combination of claim 8, wherein the organic fluorescent label is Fluorescein Isothiocyanate (FITC) and the inorganic fluorescent label is selected from fluorescent nanoparticles, Quantum Dots^TMCy3 and Cy5。

10. A combination of pathogen-specific probe DNA sequences for detecting and identifying specific pathogens causing a pathogen selected from the group consisting of infectious endophthalmitis, keratitis, uveitis, retinitis, meningitis and encephalitis, wherein said combination consists of the following probe DNA sequences:

“cgcttggtttcggatgggaggcaactgtgctatccccatcacggtcatggagtacaccgaatgctcctacaacaagtctctgggggc”(SEQ ID No.45)

“ggcaatcgtgtacgtcgtccgcacatcacagtcgcggcagcgtcatcggcggtaacgcaagacccccccg”(SEQ ID No.46)

“caagctgacggacatttacaaggtccccctggacgggtacggccgcatgaacggccggggcgtgtttcgcgtgtgggac”(SEQ ID No.47)

“ttccggctcatggcgttaaccaggtagaaactgtgtgtacagttgcgttgtgcgtaacgtaaaagcagggcg”(SEQ ID No.48)

“cggcgacgacgacgataaagaatacaaagccgcagtgtcgtccagaggattacgcgaccagattg”(SEQID No.49)

“gggcacgtcctcgcagaaggactccaggtacaccttgacgtactggtcacctatcacctgcatcttgg”(SEQ ID No.50)

“ggtcttgccggagctggtattaccttaaaactcactaccagtcatttctatccatctgtctttgtctttcacggaggca”(SEQ ID No.51)

“tccatttaacgttgcatcattttgtgttatcatagaactgcgtaaacactcggcaagtaatacagataactcgctaccggaacgt”(SEQ ID No.52)

“cgccgccaacatgctctaccctatacccgccaacgctaccaacgtgcccatatccatcccctcccgcaac”(SEQ ID No.53)

“tgggctacacacgtgctacaatggtcggtacagagggtcgccaaaccgcgaggtggagctaatctcacaaaaccgatcgtagtccg”(SEQ ID No.54)

“ggcctaacacatgcaagtcgagcggatgaaaggagcttgctcctggattcagcggcggacgggtgagtaatgcctaggaatctgcc”(SEQ ID No.55)

“acgtcaaatcatcatgcccccttatgacctgggctacacacgtgctacaatggacggtacaaagggctgca”(SEQ ID No.56)

“gcggaacgtgggaccaatacctgggttgggccggctgcttcgggcagcaactcccccgggttgaagaagaaaatcaccccgtcg”(SEQ ID No.57)

“aacttttttgactgccagacacactattgggctttgagacaacaggcccgtgccccttttggggggtggcatcc”(SEQ ID No.58)

“tggttactcgcttggtgaatatgttttataaatcctgtccaccccgtggataggtagtcggcaaaacgtc”(SEQ ID No.59)

“cccctctgctggcgaaaagtgaaattcatgagtatctgtgcaactttggtgtattcgcagattggtcgcc”(SEQ ID No.60)

“aatcgtatctcgggttaatgttgcatgatgctttatcaaatgacaagcttagatccgtttctcatacggttttcctcga”(SEQ ID No.61)

“gctgggactgaggactgcgacgtaagtcaaggatgctggcataatggttatatgccgcccgtcttgaa”(SEQ ID No.62)

“tggcgaacgggtgagtaacacgtgagtaacctgcccttgactttgggataacttcaggaaactggggctaataccgg”(SEQ ID No.63)

“cggcggcaagttcgacgacaacacctacaaggtgtccggcggcttgcacggtgtgggcgtctcggtgg”(SEQ ID No.64)

"ccaggtcggcggagaagccgaggcaggcgaggtccttcagttcgtcgcgggtcatcgggccggtgg" (SEQ ID No.65), and

“gccgccctgaccaccttcatcagcctggccggccgttacctggtgctgatgccgaacaacccgc”(SEQ IDNo.66)。

11. the combination of pathogen-specific probe DNA sequences according to claim 10, wherein the probe DNA sequences are labeled with a biotin group at the 5' end, resulting in detection by formation of a colored product; or the probe DNA sequence is labeled with a fluorescent label selected from the group consisting of organic fluorescent labels and inorganic fluorescent labels to enable detection by any equipment or microscopic method.

12. The combination of pathogen-specific probe DNA sequences according to claim 11, wherein the organic fluorescent label is fluorescein isothiocyanate FITC and the inorganic fluorescent label is selected from fluorescent nanoparticles, Quantum DotsTM, Cy3 and Cy 5.

13. A combination of target DNA sequences, wherein the sequences are selected from:

5’gcaactgtgctatccccatcacggtcatggagtacaccgaatgct 3’(SEQ ID No.67)

5’cacatcacagtcgcggcagcgtcatcggcg 3’(SEQ ID No.68)

5’tccccctggacgggtacggccgcatgaacggccgggg 3’(SEQ ID No.69)

5’aggtagaaactgtgtgtacagttgcgttgtg 3’(SEQ ID No.70)

5’aatacaaagccgcagtgtcgtc 3’(SEQ ID No.71)

5’gactccaggtacaccttgacgtactg 3’(SEQ ID No.72)

5’cttaaaactcactaccagtcatttctatccatc 3’(SEQ ID No.73)

5’ttatcatagaactgcgtaaacactcggcaagtaata 3’(SEQ ID No.74)

5’ctatacccgccaacgctaccaacgtgccca 3’(SEQ ID No.75)

5’tcggtacagagggtcgccaaaccgcgaggtggagctaa 3’(SEQ ID No.76)

5’ggatgaaaggagcttgctcctggattcagcggcggacg 3’(SEQ ID No.77)

5’gacctgggctacacacgtgctaca 3’(SEQ ID No.78)

5’ctgggttgggccggctgcttcgggcagcaactcccccgggtt 3’(SEQ ID No.79)

5’ggctttgagacaacaggcccgtgccc 3’(SEQ ID No.80)

5’tttataaatcctgtccaccccgt 3’(SEQ ID No.81)

5’aaattcatgagtatctgtgcaactttg 3’(SEQ ID No.82)

5’atgatgctttatcaaatgacaagcttagatcc 3’(SEQ ID No.83)

5’gtaagtcaaggatgctggcataatg 3’(SEQ ID No.84)

5’gcttcagcgccgtcagcgaggataac 3’(SEQ ID No.85)

5’aacacctacaaggtgtccggcggcttgcac 3’(SEQ ID No.86)

5 'cgaggcaggcgaggtccttcagttcgtcgcg 3' (SEQ ID No.87) and

5’atcagcctggccggccgttacctggtg 3’(SEQ ID No.88)。

14. use of a primer set combination as claimed in claim 1 for the preparation of reagents for use in a method for detecting a target nucleic acid of one or more than one pathogen in a sample, the method comprising:

a) a sample is obtained and the sample is,

b) (ii) extracting DNA from the sample,

c) performing a multiplex polymerase chain reaction using the primer set combination as set forth in claim 1 to obtain amplified products,

d) (ii) denaturing the amplification product(s),

e) hybridizing the denatured amplification products to one or more than one target DNA sequence, and

f) the hybridized product is detected using conventional methods.

15. The use of claim 14, wherein the pathogen causes an external eye infection, infectious endophthalmitis, conjunctivitis, uveitis, retinitis, meningoencephalitis, or meningitis.

16. The use of claim 14, wherein the target DNA sequence is selected from SEQ ID No.67, SEQ ID No.68, SEQ ID No.69, SEQ ID No.70, SEQ ID No.71, SEQ ID No.72, SEQ ID No.73, SEQ ID No.74, SEQ ID No.75, SEQ ID No.76, SEQ ID No.77, SEQ ID No.78, SEQ ID No.79, SEQ ID No.80, SEQ ID No.81, SEQ ID No.82, SEQ ID No.83, SEQ ID No.84, SEQ ID No.85, SEQ ID No.86, SEQ ID No.87 and SEQ ID No.88 or combinations thereof.

17. The use of any one of claims 14 to 16, wherein all of the primer sets can be used together in a single tube using uniform thermocycling conditions characterized by a denaturation step of 94 ℃ for 5 minutes followed by 40 cycles of 60 ℃ to 64 ℃ for 45 seconds, 72 ℃ for 45 seconds and 94 ℃ for 45 seconds, and 72 ℃ for 10 minutes of extension reaction.

18. The use of any one of claims 14-16, wherein the primer is labeled with a biotin group at the 5' end, resulting in detection by formation of a colored product; or wherein the primer is labeled with a fluorescent label selected from the group consisting of an organic fluorescent label and an inorganic fluorescent label, enabling detection by any fluorescent scanning device or microscopy method.

19. The use of claim 18, wherein the organic fluorescent label is Fluorescein Isothiocyanate (FITC) and the inorganic fluorescent label is selected from the group consisting of fluorescent nanoparticles, Quantum dots, Cy3, and Cy 5.

20. The use of any one of claims 14-16, wherein the hybridization product is detected in a macroarray, slot hybridization or linear probe assay using a specific probe DNA sequence selected from the group consisting of SEQ ID nos. 45-66 and combinations thereof.

21. Use according to claim 20, wherein the macroarray comprises the target DNA sequence SEQ ID No.67, SEQ ID No.68, SEQ ID No.69, SEQ ID No.70, SEQ ID No.71, SEQ ID No.72, SEQ ID No.73, SEQ ID No.74, SEQ ID No.75, SEQ ID No.76, SEQ ID No.77, SEQ ID No.78, SEQ ID No.79, SEQ ID No.80, SEQ ID No.81, SEQ ID No.82, SEQ ID No.83, SEQ ID No.84, SEQ ID No.85, SEQ ID No.86, SEQ ID No.87, SEQ ID No.88 or combinations thereof immobilized on a solid phase consisting of nitrocellulose, nylon, glass or polystyrene.

22. The use of claim 21, wherein the solid phase is comprised of charged nylon.

23. A kit for simultaneously detecting and differentiating all pathogens causing external eye infection, endophthalmitis, uveitis, retinitis, or meningoencephalitis, said kit comprising:

a) the set of forward and reverse primers as claimed in claim 1,

b) a matrix of DNA sequences as claimed in claim 13 immobilized on a suitable solid phase,

c) standard reagents required for amplification of DNA by polymerase chain reaction,

d) standard reagents required for hybridizing the PCR amplification product to a matrix of DNA sequences immobilized on a suitable solid phase,

e) standard reagents required for detection and identification of the final hybridization product.

24. A kit for detecting a pathogen in a sample, the kit comprising primer sets 1-22(SEQ ID nos. 1-44), wherein the pathogen is herpes simplex virus 1 and 2, cytomegalovirus, varicella zoster virus, adenovirus, eubacteria, gram negative bacteria, fungi, toxoplasma gondii, and chlamydia trachomatis.

25. The kit of claim 24, wherein the pathogen is a gram-positive bacterium.

26. The kit of claim 24, wherein the pathogen is mycobacterium tuberculosis, mycobacterium cheloniae, or mycobacterium fortuitum.

27. A set of target DNA sequences immobilized thereon, wherein said target DNA sequences have the nucleotide sequences shown as SEQ ID Nos. 67-88.