JP2006149390A - Rapid detection of isoniazid resistance in Mycobacterium tuberculosis - Google Patents

Abstract

[PROBLEMS] To know the molecular mechanism of resistance to isoniazid (INH), which is a major component in anti-tuberculosis therapy, and to provide a simple test method capable of rapidly identifying an INH-resistant strain and a nucleic acid probe and nucleic acid hybridization. A kit for performing is provided.
A method and kit for detecting by a nucleic acid hybridization or amplification method with a unique nucleic acid sequence of a chromosomal region containing a Kat G gene that mediates INH-sensitivity.
[Selection figure] None

Description

  The present application is serial number No. filed on April 30, 1992. 875,940 is a continuation-in-part application. The present invention relates to the rapid detection of Mycobacterium tuberculosis strains resistant to the antibiotic isoniazid. More specifically, the present invention relates to a method for detecting isoniazid resistance in Mycobacterium tuberculosis by nucleic acid hybridization. The invention also relates to nucleic acid probes and kits for performing nucleic acid hybridization.

  Despite more than a century of research since tuberculosis was discovered by Robert Koch as a virulence factor for tuberculosis, tuberculosis remains one of the leading causes of human morbidity and mortality. Death from tuberculosis reaches 3 million annually (Snider, 1989), most of which are in developing countries, but tuberculosis is again an important issue in the west due to the increase in homeless population and the impact of the AIDS epidemic (Non-patent document 1: Chaisson et al., 1989; Sider and Roper, 1992).

Isonicotinic acid hydrazide, or isoniazid (INH), is a group of “tuberculosis”-Mycobacterium tuberculosis, Bovine tuberculosis and M. tuberculosis. It has been used for the treatment of tuberculosis for the past 40 years because of its strong medicinal properties against africanum species (Non-patent Document 2: Middlebrook, 1952; Youatt, 1969). Although the target of INH is not precisely known and the mechanism of action is unknown, it is known that several metabolic pathways are disturbed by administration of this drug. There is a lot of evidence to show that INH appears to act as an antimetabolite of NAD and pyridoxal phosphate (Non-Patent Document 3: Bekierkunst and Bricker, 1967; Sriprapash and Ramakrishnan, 1970; Winder and Collins, 1969, 1970). In addition, there is data showing that this drug blocks the synthesis of mycolic acid, which is responsible for the acid resistance property of the tuberculosis cell wall (Non-Patent Document 4: Winder and Collins 1970; Quemard et al., 1991). . Soon after INH was used, INH-resistant Mycobacterium tuberculosis isolates appeared, and as a result of investigating the characteristics, it was found that catalase-peroxidase activity was lost, and the toxicity of guinea pigs was weakened ( Non-Patent Document 5: Middlebrook et al., 1954; Kubica et al., 1968; Sriprapash and Rama-Krishman, 1970).
Chaisson et al. , 1989; Snider and Roper, 1992 Middlebrook, 1952; Youatt, 1969 Bekierkunst and Bricker, 1967; Sriprapash and Ramakrishnan, 1970; Winder and Collins, 1968, 1969, 1970 Winder and Collins 1970; Quemard et al. 1991 Middlebrook et al. 1954; Kubica et al. , 1968; Sriprapash and Rama-Krishman, 1970

  More recently, tuberculosis epidemics in the USA due to a multidrug resistance (MDR) variant of Mycobacterium tuberculosis (CDC, 1990; 1991a, b) and these variants have been extensively observed in HIV-infected and healthcare workers. The INH-resistance problem has come to new importance since it was found to be responsible for infection (Snider and Roper, 1992). Given the importance of this problem, the link between INH resistance and catalase-peroxidase production needs to be clarified.

  More specifically, it is necessary to know the molecular mechanism involved in drug sensitivity. In addition, there is a need to develop a simple test method that can rapidly identify INH-resistant strains. Furthermore, it is necessary to make a reagent for carrying out such a test method.

Thus, the present invention helps meet the above-mentioned needs by providing a process for detecting in vitro the presence of Mycobacterium tuberculosis cells that are resistant to isoniazid. The process consists of the following steps:
(A) deposit and fix the nucleic acid of the cell so that it can be in close proximity to the probe;
(B) contacting the immobilized nucleic acid obtained from step (A) with a probe under conditions that allow hybridization;
(C) Wash the filter from step (B) to remove any unhybridized probe; then (D) all hybridized probes on the washed filter from step (C) Is detected.

The probe contains the nucleic acid sequence present in the 2.5 Kb EcoRV-KpnI fragment of plasmid pYZ56, which contains a BamHI cleavage site. This fragment has been found to associate with intracellular DNA of isoniazid-sensitive Mycobacterium tuberculosis, such that such antibiotic-sensitive microorganisms do not contain DNA that hybridizes with this fragment under the conditions described below, Can be distinguished from isoniazid-resistant Mycobacterium tuberculosis.

Furthermore, the present invention relates to the nucleotide sequence of M. tuberculosis Kat G gene conferring isoniazid susceptibility that is not present in DNA from isoniazid-resistant cells, such as nucleotide sequence of isoniazid-resistant M. tuberculosis, such as RNA and DNA. Also prepare-.
The present invention also provides a probe comprising a label, such as a nuclide, attached to the nucleotide sequence of the present invention.

The present invention further provides a hybrid double complex molecule comprising the nucleotide sequence of the present invention formed by hydrogen bonding with a nucleotide sequence of a complementary base sequence, such as DNA or RNA.
The present invention also provides a process for selecting a nucleotide sequence encoding a catalase-peroxidase gene of Mycobacterium tuberculosis, or a portion of such a nucleotide sequence, from a group of nucleotide sequences, which nucleotide sequence contains It comprises the steps of deciding whether to hybridize with the nucleotide sequence of the invention. The nucleotide sequence is either a DNA sequence or an RNA sequence. The process includes detecting a label on the nucleotide sequence.

  Furthermore, the present invention provides a kit for the detection of Mycobacterium tuberculosis resistant to isoniazid. The kit consists of a set of containers containing a probe containing the nucleotide sequence where the 2.5 Kb EcoRV-KpnI fragment of plasmid pYZ56 contains the BamHI cleavage site. The kit also includes a set of containers containing a control preparation of nucleic acids.

  The present invention also encompasses compounds obtained as enzymes catalase, or products produced by the action of similar enzymes on isoniazid. The Kat G gene or a derivative that retains similar activity can be used as a source of catalase protein. The new compound is ISBN N ° 0995-2454, H.P. edited by Pasteur Institute. Screening by reactivity against INH-resistant Mycobacterium tuberculosis using an anti-biogram method as described in David et al., "Laboratory Methods for Mycobacteria Clinical Practice".

BRIEF DESCRIPTION OF THE INVENTION The figure further to will be described in greater detail by reference to the following figures, which are as follows:
FIG. 1 (A) is a physical map of Mycobacterium tuberculosis DNA cloned in shuttle cosmid pBH4. Restriction sites for C1aI (C), NotI (N) and HindIII (H) are shown. No restriction sites have been found for DraI and AsnI, and the cosmid does not have any of the additional known genes or insertion sequences described in Mycobacterium tuberculosis. The position and scale bar of the Kat G gene are shown.

FIG. 1B is a restriction map of an insert existing in pYZ56, and shows the position of Kag and the direction of transcription.
FIG. 2 shows the partial sequence of Mycobacterium tuberculosis catalase / peroxidase polypeptide and Comparison with HPI enzyme from stearothermophilus. The same residue is indicated with *.
FIG. 3 shows detection of recombinant Mycobacterium catalase / peroxidase by activity staining. Cell extracts were separated by polyacrylamide gel electrophoresis and stained for peroxidase (lanes 1-5) and catalase activity. Samples were from tuberculosis lane 1; E. coli TG1, lanes 2, 6; TG1 / pYZ56 (Kat G ⁺ ), lanes 3 and 7; TG1 / pBAK16 (lacZ ′ :: KatG), lanes 4 and 8; TG1 / pYZ56 (Same as pYZ55 lacking the 1.4 Kb BamHI-KpnI fragment).

  FIG. 4 shows the results of Southern blot analysis of various Mycobacterium tuberculosis strains using a 4.5 Kb KpnI fragment as a probe. Genomic DNA digested with KpnI is from the H37RV strain. Lane 1, strain 12, lane 2; B1453, lane 3; strain 24, lane 4; 79112, lane 5; 12646, lane 6; Strains B1453 and 24 are resistant to high concentrations of INH, strain 12 is resistant to low concentrations of INH, while others are sensitive to INH. As a control, the same blot was hybridized with a probe for the sodA gene (Zhang et al., 1991). Note the IS6110-mediated polymorphism associated with B1453.

FIG. 5 shows INH-resistant M.P. Shows a strain of M. smegmatis, BH1 ¹¹ (mc ² -155 derivative of ¹⁰ strains) were transformed with a pool of M. tuberculosis H37RV shuttle cosmids (provided from W.R.Jacob Dr. New York), each clone INH -Counted for sensitivity. Cosmid pBH4 consistently conferred drug sensitivity and the transformant overproduced catalase (assayed in Heym). The restriction map of the DNA insert from pBH4 is shown side by side with the map of the insert from pYZ55. This pYZ55 is a plasmid containing the Kat G of Mycobacterium tuberculosis H37RV, which was isolated based on the hybridization with the following oligonucleotide probe designed to match the amino acid sequence from the conserved region of E. coli HPI. is there:

(5'-TTCATCCCGCATGGCCTGGCACGGCGCGGGGCACCTACCCGC-3 '). Restriction sites for the following enzymes are indicated: B, BamH1; C, Cal; E, EcoRV; H, Hind111, K, Kpn1; M, Smal; N, Notl; R, EcoR1; Mycobacterial shuttle plasmid containing a 4.5 Kb insert from pYZ55, PBAK14, Zhang et al . , 1991, similarly confers INH-sensitivity. MIC'S is also shown for BH1 transformed with a subfragment derived from pYZ55 and inserted into pBAK14 in one (+) or other (-) coordination. Both the Kat G gene and the ability to confer INH-sensitivity mapped to the 2.9 Kb EcoRV-Kpn1 fragment (pBAK-KE ⁺ ).

FIG. 6 shows extracts from Mycobacterium tuberculosis H37RV transformed with various plasmid structures prepared for the active gel analysis ¹⁸ described above and those from E. coli. A non-denaturing gel containing 8% polyacrylamide was stained for catalase (panel A) and peroxidase (panel B) activity as described by Wayne and Diaz ¹⁹ . Lane 1, M. tuberculosis H37RV; 2, E. coli UM2 (Kat E, Kat G, ref. 15); 3, E. coli UM2 / pYZ55; 4, E. coli UM2 / pYZ56 (pUC19 corresponding to pBAK-KE ⁺ in FIG. 1) 2.9 Kb EcoRV-Kpn1 fragment); 5, E. coli UM2 / pYZ57 (pYZ55 with BamH1-Kpn1 deletion, corresponding to pBAK-KB ⁺ in FIG. 1). Under these conditions, Mycobacterium tuberculosis catalase and peroxidase activity migrated as two bands (lane 1); the same pattern was seen for the recombinant enzyme expressed in pYZ55 (lane 3). pYZ56 (lane 4) expresses a protein of increased molecular weight by fusion between Kat G and lac Z 'from the vector shown in panel C. Panel C also shows a partial sequence sequence with E. coli HP1 (the full sequence of the gene will be published somewhere).

FIG. 7 is an E. coli strain with mutations in both Kat G and Kat E (UM2, ref. 15), which includes only the PUC19 vector (hatched bars), pYZ55 expressing Mycobacterium tuberculosis Kat G (white bars), and It is transformed with pYZ56 (black bar) that expresses a large amount of Mycobacterium tuberculosis Kat G. The overnight cultures in Luria-Bertani broth with the appropriate amount of antibiotics were plated in the presence of various concentrations of INH and the colony generating units were counted. The results of a typical experiment are shown with error bars indicating the standard deviation for triplicate samples. M. tuberculosis Kat G overexpression similarly sensitized to high concentrations of INH in E. coli UM255 (Kat G, Kat E, Mulvey et al. , 1988), but in catalase positive strains such as E. coli TG1 It did not act on it. In some experiments, high concentrations of INH showed a detectable inhibitory effect on the growth of UM2 and UM255 alone, but in all experiments, inhibition of pYZ56-transformant was greater than that observed with the corresponding vector control. Was at least 10-100 times larger.

FIG. 8 shows the cleavage with Kpn1 from different tuberculosis strains, probed with (A) Kat G (4.5 Kb Kpn1 fragment), and (B) SOD gene (1.1 Kb EcoR1-Kpn1 fragment, Zhang et al. , 1991) . 1 shows a Southern blot prepared using the genomic DNA prepared. Probe labeling and blot processing were performed as previously described ¹⁶ . Lane 1, H37RV; 2, strain 12-MIC 1.6 μg / m11NH; 3, B1453-MIC> 50 μg / ml INH ²⁰ ; 4, strain 24-MIC> 50 μg / ml INH; 5,79112-INH-sensitive ²¹ ; 6,12646-INH-sensitive ²¹ ; 7,79665-INH-sensitive ²¹ . INH sensitivity was confirmed by seeding of Lowenstein-Jensen s ops with different INH concentrations.

Detailed Description of Preferred Embodiments The emergence of a number of Mycobacterium tuberculosis strains that are recently multidrug resistant in the United States is the most surprising event, due to the unusually strong contact sensitivity of this bacterium. This risk is clearly demonstrated in several small tuberculosis epidemics, where one patient infected with MDR tuberculosis infected both HIV-positive prison guards and healthy nursing staff (CDC 1990, 1991). Daley et al., 1992; Snider and Roper, 1992). In view of the current severity of the HIV epidemic, western AIDS patients have become MDR tuberculosis strains (rather than avian M. tuberculosis / intracellular mycobacterial complexes) like African patients. If infected, it is expected that the disease will spread on a global scale.
Isoniazid (INH) is a mycobacterial group of Mycobacteria-Mycobacterium tuberculosis, Mycobacterium bovis, It is a drug having a particularly strong bactericidal action against africanum-, and has exerted special efficacy in the treatment of tuberculosis. Standard anti-tuberculosis treatment regimens generally give INH and rifampicin, but often a combination of the weaker drugs pyrazinamide, ethambutol or streptomycin is used. In addition to being used for treatment, INH is also given to people in direct contact with patients as a preventive method.

INH-resistant mutants of Mycobacterium tuberculosis, the causative agent of human disease, exhibit two levels of resistance: low (1-10 μg / ml) and high (10-100 μg / ml). INH-resistance is often accompanied by loss of catalase activity and fungal toxicity. Recently, with the epidemic of AIDS, increasing homelessness and worsening social conditions, tuberculosis has reappeared as a serious public health problem in developed countries, particularly the United States. The formidable aspect of this disease today is the emergence of multidrug-resistant bacteria and the rapid nosocomial infection of healthcare workers and HIV-infected patients. For this reason, CDC has issued new recommendations on the treatment and transmission prevention of multi-drug resistant strains (at least INH and rifampicin). The following studies were conducted to gain new perspectives on the INH-resistance problem and to develop rapid diagnostic test methods.
Naturally, what is essential is to elucidate the mechanism of resistance to the main anti-tuberculosis drugs INH and rifampicin, thereby creating a new chemotherapeutic strategy to be developed, New compounds can be planned to be suppressed.

  The present invention proves that the catalase-peroxidase enzyme, HPI is effective as described above, and considers that this enzyme alone mediates toxicity. Evidence supporting this conclusion is as follows. That is, expression of the Mycobacterium tuberculosis Kat G gene in a catalase negative mutant of E. coli makes this bacterium susceptible to INH. Furthermore, isolation of the Mycobacterium tuberculosis INH-sensitive gene, Kat G, is important in that it facilitates rapid detection of INH-resistant strains by hybridization and PCR-based approaches. The high frequency of Kat G deletions in clinical strains simplifies this procedure, as shown here.

Identification of Mycobacterium tuberculosis genes contained in INH-susceptibility One heterogeneous approach was used to isolate Mycobacterium tuberculosis genes contained in INH-susceptibility. BH1 is a spontaneous mutant of the smegma strain MC ² 155 that easily transforms (Snapper et al., 1990) and is resistant to 512 μg / ml INH and lacks catalase-peroxidase activity (Heym et al., 1992). Since there is a strict correlation between INH-sensitivity and the activity of these enzymes, transformation of BH1 by plasmids carrying the appropriate gene from Mycobacterium tuberculosis is associated with INH-resistance that coincides with their recovery. Should lead.
Therefore, DNA was prepared from a pool of M. tuberculosis shuttle cosmids in E. coli and then introduced into BH1 by electro-transformation. Over 1000 kanamycin-resistant transformants were then counted for INH-resistance, resulting in MICs from 4 clones that could not grow on medium containing 32 g / ml INH, ie wild type MC ² 155.

  After retransformation of BH1, only one of these, only pBH4, consistently conferred an INH-sensitive phenotype. It was found that the size of the chromosomal DNA carried by pBH4 by cleavage with the restriction enzymes BamHI, KpnI, NotI, ClaI and HindIII was about 30 Kb. A map generated by the last three of the above enzymes is shown in FIG.

  When pBH4 was used as a hybridization probe to detect homologous clones in the library, eight more shuttle cosmids were isolated. By transformation to BH1, five of these (T35, T646, T673, T79, T556) regained INH-resistance and showed a similar restriction profile as pBH4 (data not shown). In particular, a 4.5 Kb KpnI fragment was present in all cases.

  Attempts to subclone individual BamHI fragments did not result in a transformant that could complement and complete the injury in BH1, suggesting that the BamHI site is located in the gene of interest. It was. In contrast, pBH5, a derivative of pBH4, was created by deletion of the EcoRI fragment, indicating that a 7 Kb fragment is not required for restoration of INH-sensitivity.

BH1 of INH- complementary and which are examined carefully transformants include shuttle cosmid resistant mutant were established MIC _S for several antibiotics. In all cases, the MIC for INH decreases from 512 to 8 μg / ml, which is lower than the value of the sensitive strain MC ² 155 (32 μg / ml). Such an oversensitive phenotype suggests that the recombinant clone may have overproduced an enzyme with the ability to enhance INH-toxicity. Enzymatic studies have shown that these transformants all produce about twice as much peroxidase and catalase as the INH-sensitive wild-type MC ² 155 strain (data not shown).

Many of the MDR strains of Mycobacterium tuberculosis have lost sensitivity to not only INH but also rifampicin, streptomycin, ethambutol and pyrazinamide. To investigate the possibility that there may be a correlation between INH and resistance to these compounds, various M.P. The MIC _S of several drugs against Smegmatis strains and their pBH4 transformants was determined, but no difference was found.

Cloning of Mycobacterium tuberculosis catalase gene 45-mer oligonucleotide based on primary sequence of highly conserved region in catalase-peroxidase enzymes of E. coli (Triggs-Raine et al., 1989) and Bacillus stearothermophilus (Loprasert et al., 1988) A probe was designed. By probing a genomic blot of Mycobacterium tuberculosis DNA with this oligonucleotide, a unique band was detected in most cases. Since KpnI produced a 4.5 Kb unique fragment that hybridizes strongly, this enzyme was used to create a library that selects for size in PUC19.

  A suitable clone, pYZ56, was obtained by screening with the oligonucleotide probe. The restriction map of the inserted DNA is shown in FIG. 1, and it can be seen that this corresponds exactly to the portion of pBH4. It was also confirmed independently by cross-hybridization.

  Several subcloning experiments showed that the smallest fragment expressing Mycobacterium tuberculosis catalase-peroxidase activity in E. coli was a 2.5 Kb EcoRV-KpnI fragment, which, as expected, contained a cleavage site for BamHI. . From partial DNA sequence analysis, the Kat G gene carried by pYZ56 was found in E. coli and B. coli. It was found to encode a catalase-peroxidase enzyme that is very homologous to the stearothermophilus HPI enzyme.

Mycobacterium tuberculosis APLNSWPDNASLDKARRLWPSKKKYGKKLSW
ADLIV
Escherichia coli ************ V ********* I * Q *** Q * I ***
*** FI
B. stearo-
thermophilus ******** N ********* C * GR ***
RNT * T *-* LGPICS
(FIG. 2; Triggs-Raine et al., 1988); (Loprasert et al., 1988). The same residue is indicated by *. HPI activity was determined by staining with E. coli, It was detected in all smegmatis (see below).

Since the Mycobacterium tuberculosis Kat G gene, which contains catalase-peroxidase during INH-sensitivity, was cloned, it was an urgent concern to investigate the genetic basis of the correlation between catalase-negative and isoniazid resistance. A series of constructs was established in shuttle vector pBAK14, and INH-resistance M.I. Smegmatis mutant BH1 was used to transform. Only plasmids carrying the complete Kat G gene produced HPI and regained INH-sensitivity. The smallest of these, pBAK16, carries the 2.5 Kb EcoRV-KpnI fragment, which does not include the upstream 2 Kb region of Kat G, and is sufficient for catalase-peroxidase alone to make mycobacteria sensitive to INH. It is shown that.

  Cell-free extracts were separated by non-denaturing polyacrylamide gel electrophoresis and stained for peroxidase and catalase activity. Under these conditions, the M. tuberculosis enzyme showed two bands of peroxidase activity (lane 1), which co-migrated with catalase activity (data not shown; Heym et al., 1992).

  When the Kat G gene was introduced into E. coli, the same protein was synthesized, whereas pYZ56 produced a slightly larger protein. This is due to the construction of the in-frame lac Z :: Kat G gene fusion. M.M. Activity staining was also performed on cell extracts of Smegmatis. The presence of the Kat G gene from Mycobacterium tuberculosis in BH1 leads to the production of catalase-peroxidase enzyme, which is an enzyme made in Mycobacterium tuberculosis or E. coli, and It showed the same electrophoretic mobility as the native HPI of Smegmatis.

It has long been known that a subset of INH-resistant strains of Mycobacterium tuberculosis , particularly those resistant to the highest drug concentration, are less toxic to guinea pigs and lack catalase activity. Genomic DNA was prepared from several clinically isolated M. tuberculosis and analyzed by Southern blotting using a 4.5 Kb KpnI fragment as a probe. Of the two highly resistant strains, B1453 and 24, the catalase gene was deleted from the chromosome. On the other hand, those showing low resistance like the strain 12 still existed but not expressed. Furthermore, additional studies have shown that the region immediately before Kat G is susceptible to dislocation (data not shown).

To determine whether the M. tuberculosis HPI is E. coli HPI enzyme of M. tuberculosis to be the sensitivity can give INH susceptible to E. coli against INH, a series of catalase mutants was transformed with pYZ55 MIC _S Was quantified. Wild-type strains were not INH sensitive, but mutants containing pYZ56 showed growth inhibition in the presence of high concentrations of INH (500 μg / ml) while lacking endogenous catalase activity. On the other hand, the non-transformed strain was insensitive.

  For the purposes of the present invention, the plasmid-containing and restriction endonuclease map shown in FIG. I-1209 was deposited on May 18, 1992 in a strain of National Collection of Cultures of Microorganism, Parry, Laboratories, France. This plasmid contains the nucleic acid sequence of the present invention, ie, a 4.5 Kb KpnI-KpnI fragment of plasmid pYZ56 with a BamHI cleavage site in the fragment.

  In general, the invention features a method for detecting the presence of isoniazid-resistant Mycobacterium tuberculosis in a sample, which can be selectively hybridized with Isoniazid-resistant Mycobacterium tuberculosis DNA to form a detectable complex. It also includes providing at least one such DNA or RNA. Detection is performed under conditions that allow the probe to hybridize with Isoniazid-sensitive Mycobacterium tuberculosis DNA present in the sample to form a hybrid complex and to detect the hybrid complex as an indication of the presence of Isoniazid-sensitive Mycobacterium tuberculosis in the sample. (As used herein, “selective hybridization” refers to a DNA or RNA probe that does not form a hybrid with Isoniazid-resistant Mycobacterium tuberculosis but hybridizes only with Isoniazid-sensitive Mycobacterium tuberculosis). It can be made from Mycobacterium tuberculosis cells or parts of the cells or cell components rich in Mycobacterium tuberculosis nucleic acids, in particular DNA. Hybridization can be performed using a normal hybridization reagent. No specific hybridization conditions were found that were critical to the present invention.

More specifically, the DNA sequence from Mycobacterium tuberculosis can be analyzed by Southern blotting and hybridization. The method used in the present invention is described in Maniatis, Sambrook et al. (Cold Spring Harbor, Second Edition, 1989). The DNA fragments can be separated on an agarose gel and denatured there. The fragment is then transferred from the gel to a water-insoluble solid, ie a porous support such as a nitrocellulose filter, nylon membrane, or activated cellulose paper, where it is fixed, for example Hybond available from Amersham, for example. ^TM membrane is used. After prehybridization to reduce non-specific hybridization with the probe, the solid support is hybridized with the nucleic acid probe of the present invention. The solid support is washed to remove unbound and weakly bound probes and the resulting hybrid double complex molecule is examined. An alternative convenient approach is to hybridize the oligonucleotide to the denatured DNA in the gel.

  The amount of labeled probe present in the hybridization solution varies greatly depending on the nature of the label, the amount of labeled probe that can naturally bind to the filter and the stringency of the hybridization. In general, an excess of well over the stoichiometric amount is used to promote the rate of probe binding to the immobilized DNA.

  Hybridization with various stages of stringency can be used. The more stringent the conditions, the greater the complementarity required for hybridization between the polynucleotide and probe for duplex complex formation. The severity of hybridization can be controlled by temperature, probe concentration, probe length, ionic strength, time, etc. Conveniently, the stringency of hybridization can be changed by changing the polarity of the reaction solution. The temperature to be used is determined empirically or from well-known formulas developed for this purpose.

  Unlike Southern hybridization, where DNA fragments are transferred from an agarose gel to a solid support, the method of the invention can also be performed by oligonucleotide hybridization in a dry agarose gel. In this method, the agarose gel is dried and hybridization is performed at that position using the oligonucleotide probe of the present invention. This method is recommended when the speed and sensitivity of detection is desired. This method can be performed on agarose gels containing the Mycobacterium tuberculosis genome or cloned DNA.

  Furthermore, the method of the present invention can be carried out by transferring M. tuberculosis DNA from a polyacrylamide gel to a nylon filter by electroblotting. Electroblotting may be desirable when time is most important because electroblotting is necessarily faster than capillary blotting developed to transfer DNA from agarose gels. This method can be performed in combination with UV-crosslinking. A polyacrylamide gel containing the sample to be tested is placed in contact with a suitably prepared nylon filter. These are then placed in an electroblotting apparatus in a sandwich and then current is used to transfer the DNA from the gel onto the filter. After washing with buffer, the filter is prepared for prehybridization and then allowed to hybridize or UV-crosslink.

  The method of the present invention can be carried out using the nucleic acid probe according to the present invention for detecting Mycobacterium tuberculosis resistant to isoniazid. The probe can be detected using conventional methods.

The nucleotides of the present invention can be used as probes for the detection of nucleotide sequences in Mycobacterium tuberculosis biological samples. Polynucleotide probes can be labeled with atoms or inorganic free radicals, most commonly radionuclides, but possibly also with heavy metals. Radiolabels include ³² P, ³ H, ¹⁴ C or similar. Any radioactive label that provides a suitable signal and has a sufficient half-life can be used. Other labels include those that can act as specific binding pair members for labeled ligands and analogs, such as labeled antibodies, fluorescent materials, chemiluminescent bodies, enzymes, and antibodies. How the label is selected depends on the rate of hybridization and the effect of the label on the binding of the label to DNA or RNA. It is necessary that the label provides sufficient sensitivity to detect the amount of DNA or RNA for hybridization.

In a preferred embodiment of the invention, the probe is labeled with a radioactive isotope, such as ³² P or ¹²⁵ I, which can be incorporated into the label, for example by nick-translation.
In other desirable embodiments, when avidin is conjugated to biotin, it reacts with chemicals that allow detection of hybrid DNA complexes, such as avidin conjugated with a fluorophore. Label the probe with biotin. The presence of this fluorophore makes it possible to detect the hybrid DNA complex by fluorescence measurement; if the chemical substance that binds to biotin is a compound with a high electron density, the hybrid DNA complex can be detected by an electron microscope. In the case of antibodies, it can be detected immunologically; alternatively, one of the catalase / substrate pairs can be detected enzymatically. Prior to contacting the bacteria with the probe, the M. tuberculosis is disrupted to elute the DNA, which is then denatured and immobilized on a suitable solid DNA support, such as a nitrocellulose membrane.

  Another detection method that does not require probe labeling is the so-called sandwich hybridization method. In this assay, an unlabeled probe contained in a single-stranded vector forms a hybrid with Isoniazid-sensitive Mycobacterium tuberculosis DNA, and a labeled, single-stranded vector without a probe labels the entire hybrid complex, Hybridize with the vector containing the probe.

The sequences of the present invention were derived from dideoxynucleotide sequencing. The nucleotide sequence of the nucleotide is written in the 5 '----->3' direction. Each letter shown is in the usual nomenclature for the following nucleotides:
A Adenine G Guanine T Thymine C Cytosine

  The nucleotides of the present invention can be made by 3 '----> 5' phosphate bond formation between nucleosides using conventional chemical synthesis methods. For example, the well-known phosphodiester, phosphotriester, and phosphite triester methods, and known modifications of these methods can be used. Deoxyribonucleotides can be prepared on automated synthesis machines, such as those based on the phosphoramidite approach. Oligo- and polyribonucleotides can also be obtained using conventional methods and with the aid of RNA ligase.

  The nucleotides of the present invention are in a pure state. For example, these nucleotides do not include proteins derived from human blood, human serum proteins, viral proteins, nucleotide sequences encoding these proteins, human tissues, and human tissue components. Furthermore, it can be said that these nucleotides do not contain other nucleic acids, foreign proteins and lipids, and foreign microorganisms such as bacteria and viruses.

Of course, the present invention also includes variants of the nucleotide sequences of the present invention or serotype variants that exhibit the same selective hybridization properties as the probes of the present invention.
The nucleotide sequences of the present invention can be used in a DNA amplification process known as the polymerase chain reaction (PCR). For example, S.W. Kwok et al. , J .; Virol. 61: 1690-1694 (1987). PCR is advantageous because it is rapid.

A DNA primer pair of known sequence, located 10-300 base pairs away from the portion complementary to the plus and minus strands of the DNA to be amplified, is prepared by well-known methods for oligonucleotide synthesis be able to. One end of each primer can be extended and modified to create a restriction endonuclease site when the primer anneals to PBMC DNA. What the PCR reaction mixture can contain is PBMC DNA, a DNA primer pair, four deoxyribonucleoside triphosphates, MgCl ₂ , DNA polymerase and normal buffer. DNA is amplified in many cycles. In general, it is possible to increase the detection sensitivity by increasing the number of cycles. In each cycle, the temperature is raised to denature PBMC DNA for a short time, the reaction solution is cooled, and then polymerized with DNA polymerase. Consists of.

Amplified sequences can be detected using a method called oligomer restriction (OR). R. K. Saiki et al. Bio / Technology 3: 1008-1012 (1985) and SSCP PNAS 1985, Vol. 86, p. See 2766-2770. For example, after amplification, a portion of the PCR reaction mixture is separated and hybridized with an end-labeled nucleotide probe, such as a ³² P-labeled adenosine triphosphate end-labeled probe. In OR, the end-labeled oligonucleotide probe hybridizes to the amplified sequence region in solution and reconstitutes a specific endonuclease site in the process. Thus, hybridization of the labeled probe and amplified Kat G sequence results in a double stranded DNA form that is sensitive to selective restriction enzyme cleavage. After restrictive cleavage with endonuclease, the resulting sample is analyzed on a polyacrylamide gel, resulting in an autoradiogram of a portion of the gel with diagnostically labeled fragments. The presence of a Kat G sequence in PBMC _s when a diagnostic fragment (eg, 10-15 bases in length) is found in the autoradiogram.

  Since RNA may be used instead of chromosomal DNA as an original template, there is a possibility of increasing the detection sensitivity. Therefore, in the present invention, an attempt was made to use an RNA sequence complementary to the DNA sequence described herein. RNA can be converted to complementary DNA by reverse transcriptase and then amplified.

Experimental method
Bacterial strains and plasmids Table 1 outlines the bacterial strains and plasmids used in the present invention.

  The Mycobacterium tuberculosis H37RV genomic library was constructed in the shuttle cosmid pYUB18 (Snapper et al., 1988) and Dr. W. R. Courtesy of Jacobs. Other shuttle vectors used are pYUB12 (Snapper et al., 1988) and pBAK14 (Zhanget al., 1991).

Antibiotics using microbiological methods and enzymology , growth conditions, enzymology and MIC quantification are described in Heym et al. , (1992).

Methods for Nucleic Acids Standard protocols were used for subcloning, Southern blotting, DNA sequencing, oligonucleotide biosynthesis, etc. (Maniatis et al., 1989; Eiglmeier et al., 1991).

The preparation of cell-free extracts of activity-stained E. coli and mycobacteria has recently been described (Heym et al., 1992; Zhang et al., 1991). Native protein samples were prepared by polyacrylamide gel electrophoresis as described by Laemmli (1970): with the following differences from Laemmli. That is, SDS was removed from all buffers, the sample was not boiled, and β-mercaptoethanol was not included in the sample buffer. A 50-100 μg protein sample was electrophoresed on a 7.5% polyacrylamide gel, and the gel was immersed in 3 mM H ₂ O ₂ for 20 minutes with gentle stirring to detect catalase activity. When equal amounts of 2% ferric chloride and 2% potassium ferric cyanide are added, a sharp band showing catalase activity appears upon light irradiation. Peroxidase activity is detected as a brown band after soaking the gel in a solution containing 0.2-0.5 mg / ml diaminobenzidine and 1.5 mM H ₂ O ₂ for 30-120 minutes.

  The most appropriate method for making highly toxic compounds is to perinoxidatively activate INH with Mycobacterium tuberculosis HPI enzyme (Youatt, 1969; Gayathri-Devi et al., 1975). Now that the kat G gene has been isolated and characterized, it should be possible to make new derivatives of INH that can be activated in the same way.

Reference literature

A restriction map of the insert present in pYZ56 is shown. A partial sequence of Mycobacterium tuberculosis catalase / peroxidase polypeptide and a comparison with the HPI enzyme is shown. The expression of recombinant tuberculosis peroxidase / catalase is shown. Figure 3 shows the deletion of the catalase gene in the INH-resistant strain. Various restriction enzyme maps are shown. An extract from M. tuberculosis H37RV and an extract from E. coli are shown. An extract from M. tuberculosis H37RV and an extract from E. coli are shown. An extract from M. tuberculosis H37RV and an extract from E. coli are shown. It is a graph which shows the number of colonies. The result of Southern blot analysis is shown.

Claims (6)

  A purified polynucleotide comprising the base sequence of 121 to 360 shown in FIGS. 6B and 6C.   The polynucleotide according to claim 1, wherein the polynucleotide is labeled with at least one label selected from the group consisting of a radioactive label, an enzyme label, a fluorescent label, and a phosphorescent label.   A composition for detecting INH-sensitive tuberculosis bacteria comprising the polynucleotide according to claim 1 or 2.   A kit for detecting INH-sensitive Mycobacterium tuberculosis comprising the polynucleotide according to claim 1 or 2 or the composition according to claim 3. A hybrid obtained by contacting a nucleotide derived from Mycobacterium tuberculosis with a probe comprising the polynucleotide according to claim 1 or 2 under conditions that allow the probe to hybridize with the DNA of the INH-sensitive Mycobacterium tuberculosis present in the sample. Forming a complex;
Detecting the hybrid complex to reveal the presence of INH-sensitive tuberculosis in the sample. A biological sample of M. tuberculosis is contacted with a probe comprising the polynucleotide of claim 1 or 2 under conditions that allow the probe to hybridize with the KatG gene of M. tuberculosis to form a hybrid complex. When,
Detecting the hybrid complex and revealing the presence of the catalase-peroxidase gene in the sample, in a biological sample of Mycobacterium tuberculosis encoding the catalase-peroxidase gene or a part thereof A method for detecting a nucleotide sequence.