TWI392740B - Method for identifying mycobacterium - Google Patents

Description

Method for identifying mycobacteria

The present invention relates to a method for identifying Mycobacterium spp.; in particular, the present invention relates to a method for identifying mycobacteria using a specific probe.

The tuberculosis caused by Mycobacterium tuberculosis is about 9.2 million new cases each year and causes about 1.7 million deaths. Taiwan has 14,500 to 16,500 cases of tuberculosis every year, which is a major public health problem worldwide.

Mycobacteria include absolute pathogenic bacteria, facultative pathogens, and saprophytic bacteria. Nearly 100 mycobacteria have been isolated, of which more than 20 are related to human diseases. Clinically common such as Mycobacterium tuberculosis comples (MTBC), M. leprae , and ulcers M. ulcerans , M. abscessus , and M. avium complex, etc. (Murray et al., 2005, Medical Microbiology, fifth ed, vol. Elsevier Mosby.) .

The Mycobacterium tuberculosis group includes the following species: Mycobacterium africanum , M. bovis BCG, M. bovis , M. caprae , and Mycobacterium tuberculosis ( M. canettii ), M. microti and M. tuberculosis (Djelouadji et al., 2008, PLoS Negl. Trop. Dis. 2: e253; Murray et al., 2007, Manual of CLINICAL MICROBIOLOGY, 9 ^th ed, vol. 1) These strains are similar in shape to each other and cause human or animal tuberculosis. Among them, tuberculosis, tuberculosis and tubercle bacilli in Africa cause tuberculosis in humans more often (Singleton and Sainsbury, 2006). , Dictionary of Microbiology and Molecular Biology, 3 ^rd ed, vol.), especially Mycobacterium tuberculosis.

Correct identification of strains in MTBC contributes to the epidemiological investigation of tuberculosis, but the genetic similarity of MTBC members is very high (Sreevatsan et al., 1997, Proc. Natl. Acad. Sci. USA 94:9869-9874 ), there is 99-100% sequence similarity in the 16S rRNA gene, so the sequence of this gene is not easy to distinguish these species; in recent years, the target genes used to distinguish MTBC include pncA , oxyR , mtp40 , hupB , gyrB Et al. (Liebana et al., 1996, J. Clin. Microbiol. 34: 933-938; Prabhakar et al., 2004, J. Clin. Microbiol. 42: 2724-2732; Scorpio and Zhang, 1996, Nat. Med. 2: 662-667; Sreevatsan et al., 1996, J. Clin. Microbiol. 34: 2007-2010), in which the gyrB gene has been shown to be a better gene for distinguishing MTBC species (Chimara et al., 2004, Mem. Inst. Oswaldo. Cruz. 99: 745-748; Goh et al., 2006, Mol. Cell. Probes. 20: 182-190; Niemann et al., 2000, J. Clin. Microbiol. 38: 3231-3234).

Mycobacteria other than Mycobacterium tuberculosis are generally referred to as nontuberculous mycobacteria (NTM). Most NTMs are found in natural soil or water, and a few can cause opportunistic infections in humans or animals. (Field and Cowie, 2006, Chest. 129: 1653-1672), which is more susceptible to infection when the immunity is low. In recent years, the clinical separation rate of NTM has gradually increased (Griffith, 2007, Curr. Opin. Infect Dis. 20:198-203). Since NTM cannot be distinguished from Mycobacterium tuberculosis under the microscope, it is usually only after culture and isolation that strain identification can be performed, so it takes a long time to identify. Today there are some methods molecule binding techniques, for example: the use of restriction fragment length diversity IS 6110 (IS 6110 -restriction fragment length polymorphism, RFLP) (Kallenius et al., 1999, J Clin Microbiol 37: ... 3872-3878) , heat shock protein 65 polymerase chain reaction and restriction enzyme assay ( hsp 65 PCR-restriction assay) (Telenti et al, 1993, J. Clin. Microbiol. 31: 175-178), multilocus variable number tandem repeats Analysis (mycobacterial interspersed repetitive-unit-variable-number tandem-repeat, MIRU-VNTR) or spacer spoligotyping (Aranaz et al., 2004, J. Clin. Microbiol. 42: 5388-5391; Cadmus et al., 2006, J. Clin. Microbiol. 44:29-34; Godreuil et al., 2007, J. Clin. Microbiol. 45:921-927; Kremer et al., 2005, J. Clin. Microbiol. 43:314-320; Kulkarni et al., 2005, Res. Microbiol. 156:588-596; Sola et al., 2003, Infect. Genet. Evol. 3:125-133), but sometimes not clinically Further identification of NTM.

Due to the slow growth of mycobacteria, it is generally clinically necessary to take 2 weeks to 8 weeks to identify mycobacteria, which takes a long time. In recent years, many molecular bioassays and kits have been developed, such as INNO-LiPAMYCOBACTERIA (Innogenetics, Ghent, Belgium), using rRNA operon of mycobacteria as a target sequence (Suffys et al., 2001, J. Clin. Microbiol. 39 : 4477-4482; Tortoli et al, 2003, J. Clin. Microbiol. 41: 4418-4420; Trombert-Paolantoni et al, 2004, Pathol. Biol. (Paris) 52: 462-468), can distinguish between Mycobacterium tuberculosis Group with 16 non-tuberculous mycobacteria; GenoType Mycobacterium CM/AS (Hain Lifesience GmbH, Nehren, Germany) used the 23S rRNA gene as a target for its identification (Makinen et al., 2006, Clin. Microbiol. Infect. 12:481-483; Richter et al., 2006). , J. Clin. Microbiol. 44:1769-1775), can distinguish between Mycobacterium tuberculosis and 14 species ( Mycobacterium CM) or 16 species ( Mycobacterium AS) non-tuberculous mycobacteria; Gen-Probe Amplified Mycobacterium tuberculosis direct test (Gen-Probe, Inc., San Diego, CA, USA) for detection of Mycobacterium tuberculosis using 16S rRNA as the target (Chedore and Jamieson, 2002, Int J. Tuberc. Lung Dis. 6: 913-919; Lemaitre et al., 2004, J. Clin. Microbiol. 42: 4307-4309; Sloutsky et al., 2004, J. Clin. Microbiol. 42: 1547-1551) ; the ^{Amplicor TM nucleic acid amplification test (Roche} Diagnostic Systems, Inc., Branchburg, NJ, USA) using the detection of Mycobacterium tuberculosis 16S rRNA as the target group (Bergmann and Woods, 1996, J Clin Microbiol 34 :... 1083- 1085; Michos et al., 2006, Diagn. Microbiol. Infect Dis. 54: 121-126). LCX MTB assay, ABBOTT LCx probe system (Abbott Laboratories, Abbott Park, Ill.) (Rohner et al., 1998, J. Clin. Microbiol. 36: 3046-3047) detection of Mycobacterium tuberculosis using a ligase linkage reaction; BD ProbeTec Energy transfer (ET) system (DTB) (Becton Dickinson Biosciences Microbiology Products, Sparks, MD, USA) (Barrett et al, 2002, J. Med. Microbiol. 51: 895-898) targeting IS 6110 and 16S rRNA genes Detection of Mycobacterium tuberculosis.

At present, there are many studies in which the liquid medium BACTEC MGIT 960 positive reaction culture and confirmation of acid-fast staining is positive, and then combined with molecular biological methods to identify the mycobacteria. Comprising rpoB gene using double-nested PCR and restriction enzyme analysis (rpo B duplex PCR-restriction enzyme analysis), a photoreactive biochemical reactions and Two, distinguishable 29 kinds of NTM (Shen et al., 2009, Int. J. Tuberc. Lung Dis. 13:472-479). It has also been studied to use the MGIT-positive reaction tube positive for acid-fast staining, and the PCR reaction with IS 6110 as the target was successfully carried out to identify MTBC. The detection time for positive and negative anti-acid staining of sputum smear was shortened to 6.4 days and 14.3 days, respectively (Sun et al., 2009, J. Formos. Med. Assoc. 108: 119-125). It was previously mentioned that PCR was used to amplify hsp65 and combined with RFLP technology to identify mycobacteria. Although this technique has been widely used, it still takes a lot of time in the culture process. If the decontaminated clinical specimen is directly digested, it is often The sensitivity is reduced due to insufficient bacteria. Chang et al. combined capillary electrophoresis and nested polymerase chain reaction (nested-PCR) to increase the sensitivity of hsp65 and RFLP (Chang et al, 2008, Talanta. 77: 182-188). Wu et al., from Chang Gung Hospital, Linkou, Taiwan, showed that with hsp65 as the target, it was good to detect MTBC and some NTM in positive smear samples by nested-PCR and RFLP (Wu et al., 2008, J. Clin). Microbiol. 46:3591-3594).

However, the above methods have many disadvantages such as complicated operation and long time-consuming, and many methods cannot identify the species, and the accuracy and sensitivity are not satisfactory. The industry still needs to develop a method for correctly identifying mycobacteria.

It is an object of the present invention to provide a method for identifying mycobacteria, which comprises subjecting a probe of Myc2 and Myc3 or a complementary strand thereof to a heterozygous reaction with DNA in a sample to be tested, if at least one is heterozygous When the reaction occurs, the sample to be tested contains mycobacteria.

Another object of the present invention is to provide a probe comprising a group selected from the group consisting of the following probes and their complementary strands: Myc2, Myc3, MTBC2, MTBC4, MbovG1, MbovGW1, Mbov1, MbovW1, Mcan1, McanW1, Mcap1 McapW1, Mmic1, MmicW1, Mtub1R, MtubW1R, Mabs1, Mavi1, Mche3, Mfor1, Mgas2, Mgor1, Mint2, Mkan1, Msi/le1, Mma/ul2, Mnon1, Mper1, Mscr1, Msme3, Mszu1, Mter1 and Mxen2.

It is still another object of the present invention to provide a kit for identifying mycobacteria in a sample comprising the aforementioned probe.

The correct rate of identification according to the method of the invention is quite high, the identification time is shortened, the identification process is consistent, and the simplification, rapidization, and automation are easy to be performed, and can be clinically applied by correct strain identification. Correct antibiotic treatment.

The object of the present invention is to provide a method for identifying mycobacteria, which comprises subjecting a probe of Myc2 and Myc3 or a complementary strand thereof to a heterozygous reaction with DNA in a sample to be tested, if at least one heterozygous reaction occurs, The sample to be tested contains mycobacteria.

The probe information used in the present invention is shown in Table 1.

The specific probe naming method is to take the uppercase English letter "M" of the genus genus and the first three lowercase English letters of the species, and finally add the serial number of the probe. The anti-sense probe will be preceded by the capital letter R. There are 19 probes selected from the rRNA intergenic spacer region (ITS); the other 12 probes are used to distinguish the strains in the MTBC, and the single nucleotides unique to the gyrB gene of each species are designed. Single nucleotide polymorphism (SNP), which consists of two probes, one is a specific probe, named as described above, and the other is a probe of other MTBC species other than the target species. W to make a difference.

The probe design principle is that the sequence of the probe is infrequently mutated and:

1. The length of the probe is between 17 and 27 nucleotides, which is the most ideal range. If the probe is too short, it will cause difficulty in binding to the target DNA, and too long will cause non-specific heterozygous reaction;

2. The ratio of G+C is between 40% and 60% to reduce the probability of secondary structure generation;

3. The probe T _m value (melting temperature) at the design temperature is between heterozygous hybrid reactor (Hybridization) of (hybridization temperature) ± 5 ℃ between possible. For example, if the hybrid temperature is set at 50 ° C, the T _m value of the probe should be as much as possible 45 ° C ~ 55 ° C;

4. Try to avoid hairpin loops, palindromes or repeats.

5. Design the difference point in the middle of the probe and add 8-10 thymines at the 3' end of the probe to increase the binding of the probe to the nylon membrane;

The designed probes were searched by BLAST to determine that they were not similar to the sequences of other strains on GenBank, avoiding cross hybridization.

As used herein, the term "probe" refers to a molecule comprising at least 8 nucleotides in succession, preferably 10 to 50 nucleotides in length, more preferably 15 to 40 nucleotides in length, optimal. It is 17 to 27 nucleotides in succession. On the other hand, preferably, the 3' end of the probe comprises 8 to 10 thymines. The probe can be hybridized with the target DNA under heterozygous conditions. Those of ordinary skill in the art to which the present invention pertains can determine the conditions of the hybridization reaction, wherein preferred conditions for the hybridization reaction are carried out at a temperature of from about 40 ° C to about 65 ° C.

The term "complementary strand" as used herein refers to a nucleic acid molecule which can be hybridized to a probe according to the invention, preferably a nucleic acid molecule which is fully complementary to the base of the probe according to the invention.

The probe according to the present invention can be used to identify mycobacteria. Since mycobacteria generally grow slowly, it takes a long time to use conventional culture methods, and some mycobacteria are difficult to identify. Previous studies have pointed out that the bacterial 16S rRNA and 23S rRNA two gene spacers are species-specific in sequence, and sometimes more distinguishable to the species level than 16S rRNA gene sequencing. Moreover, the rRNA gene spacer sequence has little variability among different strains within the species (Gurtler and Stanisich, 1996, Microbiology. 142 (Pt 1): 3-16). Therefore, the present invention mainly utilizes the rRNA gene spacer as a branch. The target of bacillus identification.

Preferably, according to the method of the present invention, the Mycobacterium tuberculosis group and the non-tuberculous mycobacterial group can be further distinguished, and the probe further comprising a probe using MTBC2 and MTBC4 or a complementary strand thereof is subjected to a heterozygous reaction with the DNA in the sample to be tested, If the DNA in the sample to be tested simultaneously generates a hybrid reaction with the probe of MTBC2 and MTBC4 or its complementary strand, the sample to be tested contains Mycobacterium tuberculosis; if the DNA in the sample to be tested is not simultaneously with MTBC2 and MTBC4 The probe or its complementary strands produce a heterozygous reaction, and the sample to be tested comprises non-tuberculous mycobacteria.

More preferably, according to the method of the present invention, the species name of the strain to be identified which has been identified as Mycobacterium tuberculosis can be further identified. Since the rRNA gene spacer of the Mycobacterium tuberculosis group has 100% similarity, MTBC has multiple single-site nucleotide polymorphisms in the gyrB gene to identify different strains in the group.

Preferably, the method according to the present invention further comprises using a probe of Mtub1R and MtubW1R or a complementary strand thereof to carry out a hybrid reaction with the DNA in the sample to be tested, for example, the DNA in the sample to be tested is heterozygously reacted with Mtub1R or its complementary strand. The intensity is greater than the intensity of the heterozygous reaction of the DNA in the sample to be tested with MtubW1R or its complementary strand, and the sample to be tested comprises M. tuberculosis .

Preferably, the method according to the present invention further comprises using a probe of MbovG1 and MbovGW1 or a complementary strand thereof to carry out a heterozygous reaction with the DNA in the sample to be tested, such as the heterozygous reaction of the DNA in the sample to be tested with MbovG1 or its complementary strand. The intensity is greater than the intensity of the heterozygous reaction of the DNA in the sample to be tested with MbovGW1 or its complementary strand, and the sample to be tested comprises the M. bovis group. Preferably, the Bovine tuberculosis population comprises M. bovis , M. bovis BCG and M. caprae . In a preferred embodiment of the invention, individual strains of the Mycobacterium tuberculosis population can be identified. Preferably, the method according to the present invention further comprises using a probe of Mbov1 and MbovW1 or a complementary strand thereof to carry out a hybrid reaction with the DNA in the sample to be tested, such as DNA in the sample to be tested and Mbov1 or its complementary strand. The intensity of the reaction is greater than the intensity of the heterozygous reaction of the DNA in the sample to be tested with MbovW1 or its complementary strand, and the sample to be tested comprises Bovine M. tuberculosis or Bovine M. tuberculosis BCG; according to the method of the invention, preferably The probe comprising Mcap1 and McapW1 or a complementary strand thereof is subjected to a heterozygous reaction with the DNA in the sample to be tested, and the intensity of the hybridization reaction between the DNA in the sample to be tested and Mcap1 or its complementary strand is greater than the sample to be tested. The strength of the hybridization reaction between the DNA and McapW1 or its complementary strands, the sample to be tested contains K. tuberculosis.

Preferably, the method according to the present invention further comprises using a probe of Mmic1 and MmicW1 or a complementary strand thereof to carry out a hybrid reaction with the DNA in the sample to be tested, such as DNA in the sample to be tested and Mmic1 or its complementary strand. The intensity of the reaction is greater than the intensity of the heterozygous reaction of the DNA in the sample to be tested with MmicW1 or its complementary strand, and the sample to be tested comprises M. microti .

Preferably, the method according to the present invention further comprises using a probe of Mcan1 and McanW1 or a complementary strand thereof to carry out a hybrid reaction with the DNA in the sample to be tested, such as DNA in the sample to be tested and Mcan1 or its complementary strand. The intensity of the reaction is greater than the intensity of the heterozygous reaction of the DNA in the sample to be tested with McanW1 or its complementary strand, and the sample to be tested comprises M. canettii .

Preferably, the method according to the present invention further comprises performing a hybrid reaction with the DNA in the sample to be tested using the first group probe or its complementary strand and the second group probe or its complementary strand, such as in the sample to be tested The intensity of the heterozygous reaction between the DNA and the second group of probes or their complementary strands is greater than the intensity of the heterozygous reaction of the DNA in the sample to be tested with the first group of probes or their complementary strands, and the sample to be tested comprises Africa. Mycobacterium tuberculosis ( M. africanum ), wherein the first group of probes comprises Mtub1R, MbovG1, Mbov1, Mcap1, Mmic1 and Mcan1; and the second group of probes comprises MtubW1R, MbovGW1, MbovW1, McapW1, MmicW1 and McanW1.

On the other hand, more preferably, the method according to the present invention can further identify the species name of the strain to be identified which has been identified as non-tuberculous mycobacteria, which is mainly carried out using a probe designed by the rRNA gene spacer.

According to the method of the present invention, it is preferred to further comprise one or more selected from the group consisting of Mabs1, Mavi1, Mche3, Mfor1, Mgas2, Mgor1, Mint2, Mkan1, Msi/le1, Mma/ul2, Mnon1, Mper1, Mscr1, Msme3, Mszu1, Mter1, Mxen2 and their complementary strand probes are heterozygously reacted with the DNA in the sample to be tested; if the DNA in the sample to be tested is heterozygous with the Mabs1 probe or its complementary strand, then The test sample comprises M. abscessus ; if the DNA in the sample to be tested is heterozygous with the Mavi1 probe or its complementary strand, the sample to be tested comprises M. avium . If the DNA in the sample to be tested is heterozygous with the Mche3 probe or its complementary strand, the sample to be tested comprises M. chelonae ; such as the DNA in the sample to be tested and the Mfor1 probe Or the complementary strand thereof produces a heterozygous reaction, and the sample to be tested comprises M. fortuitum ; if the DNA in the sample to be tested is heterozygous with the Mgas2 probe or its complementary strand, the test is to be tested stomach sample comprising Mycobacterium (M. gastri); if the test sample DNA and the probe Mgor1 Unit generates complementary hybrid reactor, the Gordon test sample comprising Mycobacterium (M. gordonae); such that the test sample or the complementary DNA probe shares with Mint2 producing a hybrid reactor, the test The sample comprises M. intracellulare ; if the DNA in the sample to be tested is heterozygous with the Mkan1 probe or its complementary strand, the sample to be tested comprises M. kansasii ; If the DNA in the sample to be tested is heterozygous with the Msi/le1 probe or its complementary strand, the sample to be tested comprises M. simiae or M. lentiflavum ; The DNA in the sample to be tested is heterozygously reacted with the Mma/ul2 probe or its complementary strand, and the sample to be tested comprises M. marinum or M. ulcerans ; The DNA in the sample to be tested is heterozygously reacted with the Mnon1 probe or its complementary strand, and the sample to be tested comprises M. nonchromogenicum ; such as the DNA in the sample to be tested and the Mper1 probe or The complementary strands produce a heterozygous reaction, and the sample to be tested contains Mycobacterium phlei ( M. peregri) NUM); such that the test sample or the complementary DNA probe shares with Mscr1 producing a hybrid reactor, the test sample comprises scrofulous Mycobacterium (M. scrofulaceum); if the test sample of DNA with Msme3 probe shares producing a hybrid or its complementary reaction, the test sample contains Mycobacterium smegmatis (M. smegmatis); such that the test sample or the complementary DNA probe shares with Mszu1 produce heterozygous reactions, Then, the sample to be tested comprises M. szulgai ; if the DNA in the sample to be tested is heterozygous with the Mter1 probe or its complementary strand, the sample to be tested comprises Mycobacterium smegmatis ( M And terrae ; and if the DNA in the sample to be tested is heterozygous with the Mxen2 probe or its complementary strand, the sample to be tested comprises M. xenopi .

The sample to be tested according to the present invention may be a culture containing the bacteria to be identified, a sample taken from the patient, or a sample from the culture to be identified or further processed from the patient to obtain a DNA information therein, wherein the patient's sample The sample is preferably sputum or blood, and the method of obtaining DNA information is preferably amplified by a polymerase chain reaction.

Since the method of the present invention is identified by a probe designed by the rRNA gene spacer or the gyrB gene, the DNA in the sample to be tested preferably comprises the rRNA gene spacer or the gyrB gene of the strain to be identified; more preferably, the method The DNA in the sample to be tested contains a fragment amplified by a polymerase chain reaction. In a preferred embodiment of the present invention, the DNA in the sample to be tested uses sp1 (SEQ ID NO. 36) and sp2 (SEQ ID NO. 37) primers to amplify the rRNA gene spacer region of the strain to be identified; Preferably, the gyrB gene of the strain to be identified in the sample is obtained by amplification of Gb1f (SEQ ID NO. 38) and Gb1r (SEQ ID NO. 39) primers.

In a preferred embodiment of the invention, the DNA in the sample can be labeled. Methods of labeling DNA are well known to those of ordinary skill in the art to which the invention pertains. For example, when performing polymerase chain reaction amplification, the primer comprises a marker, preferably the marker is digoxigenin, or the labeled dUTP can be used to introduce a marker into the product.

In a preferred embodiment of the invention, the method further comprises a positive control step. Any identifying probe for identifying a known microorganism is suitable for this positive control step.

Preferably, the hybridization reaction according to the present invention can be carried out on a microarray wafer comprising a substrate and the probe is applied to the substrate. Substrate materials and coating methods have been well established in the art, wherein the substrate is preferably nylon or glass.

The probes according to the present invention are preferably arranged on the substrate in a microarray manner to allow identification of a plurality of microorganisms in a single operation, saving a lot of time, cost, space and labor. Any of the commonly used gene expression detection methods in the art can be used in the present invention, which can replace the different biochemical reactions for different mycobacteria in the prior art to identify the species. In a specific embodiment of the invention, the mycobacteria are identified by an oligonucleotide array comprising six bacteria belonging to the M. tuberculosis complex. The probe was designed from the inner transcribed region of the 16S-23S rRNA gene of the bacterium and the gyrB gene, and the probe was spotted on a nylon membrane by a micro-matrix dot machine, and the PCR product identifying the digoxigenin was used. After the hybridization reaction, the result of the reaction is exhibited by an antibody labeled with a phosphate decomposing enzyme.

The wafer first tests the type strain and the reference strain, further tests the clinical strain, and finally directly tests the clinical specimen to evaluate the feasibility of the wafer for identifying (or detecting) mycobacteria. The present invention co-tested 25 target reference strains, including 6 MTBC (15 strains) (Table 2) and NTM 19 strains (73 strains) (Table 3), a total of 88 strains. The target clinical strains were also tested, among which MTBC 80 strains (Table 2) and NTM 121 strains, a total of 201 strains (Table 3).

The wafer according to the present invention can complete the identification of the bacteria within 8 hours, and the reagents are used in a small amount, the cost is low, and the sensitivity and specificity are high. Therefore, the microarray wafer of the present invention can quickly and correctly identify different mycobacterial species and is an effective detection tool.

Another object of the present invention is to provide a probe comprising a group selected from the group consisting of Myc2, Myc3, MTBC2, MTBC4, MbovG1, MbovGW1, Mbov1, MbovW1, Mcan1, McanW1, Mcap1, McapW1, Mmic1, MmicW1 Mtub1R, MtubW1R, Mabs1, Mavi1, Mche3, Mfor1, Mgas2, Mgor1, Mint2, Mkan1, Msi/le1, Mma/ul2, Mnon1, Mper1, Mscr1, Msme3, Mszu1, Mter1, Mxen2 and their complementary strands.

It is still another object of the present invention to provide a kit for identifying mycobacteria in a sample comprising the aforementioned probe.

According to the invention, the kit further comprises reagents for obtaining the rRNA gene spacer or the gryB gene. The composition of the reagents is also well known to those of ordinary skill in the art to which the invention pertains. In a preferred embodiment of the invention, the reagents are used in a polymerase chain reaction. In a more preferred embodiment of the invention, the kit further comprises sp1 and sp2 primers and/or Gb1f and Gb1r primers. In another preferred embodiment of the invention, the kit further comprises a reagent for hybrid reaction.

The invention is illustrated by the following examples, which are not intended to limit the invention to the invention.

Example: Experimental strains, strain culture, and strain DNA

The reference strains used in this study were from American ATCC (American Type Culture Collection, Manassas, Virginia, USA), Taiwan BCRC (Bioresources Collection and Research Center, Food Industry Research and Development Institute, Hsinchu, Taiwan), and Swedish CCUG (Culture Collection). , University of G Teborg, G Teborg, Sweden), French CIP (Collection of the Institute Pasteur, rue du Docteur Roux, Paris, France), Japan JCM (Japan Collection of Microorganism, RIKEN BioResource Center, Saitama, Japan), British NCTC (National Collection of Type Cultures, Central Public Health Laboratory, London, UK).

The target reference strains used in this study have a total of 25 (88 strains) of mycobacteria, including 6 MTBC (15 strains) (Table 2), and NTM (19 strains) (Table 3); The strains (clinical strains), including MTBC 80 strain (Table 2) and NTM 121 strain, a total of 201 strains (Table 3). Among the nontarget species, there were 37 species (38 strains) belonging to the genus Mycobacterium, and 35 strains (35 strains) of other bacteria (Table 4).

All strains were cultured according to the recommended medium for each strain center (eg BBL ^TM 7H11 Agar and L) wenstein-Jensen (LJ) agar slants) and culture conditions for secondary culture.

DNA extraction

The strain DNA extraction method is a boiled method (Millar et al., 2000, J. Microbiol. Methods. 42: 139-147): sterilized water is added to a microcentrifuge tube (Eppendorf), and the colonies on the medium are suspended in the medium. The sterilized water was heated to 100 ° C in a dry bath, and after 30 minutes, it was centrifuged at 12,500 rpm for 20 minutes. The supernatant contained bacterial DNA and stored at -70 °C.

Increase and sequence of rRNA gene spacer and gyrB gene

Amplification of mycobacteria by primers sp1 (5'-ACCTC CTTTC TAAGG AGCAC C-3', SEQ ID NO. 36) and sp2 (5'-GATGC TCGCA ACCAC TATCC A-3', SEQ ID NO. 37, respectively) rRNA gene spacer (Roth et al., 2000, J. Clin. Microbiol. 38: 1094-1104); primer Gb1f (5'-TGGTT AACGC GCTAT CCAC-3', SEQ ID NO. 38) and Gb1r (5'- ACCAA CTCTC GTGCC TTAC-32, SEQ ID NO. 39) was used to amplify the gyrB gene of mycobacteria, and the primers were all synthesized by MD Bio Inc., Taipei, Taiwan. These two pairs of primers were used to increase the concentration by polymerase chain reaction, and the amplified products of the primer pair Sp1 and Sp2 were rRNA gene spacers. The product of the amplification of Gb1f and Gb1r by the primer is a partial sequence of the gyrB gene. PCR was performed with an Applied Biosystems 2720 thermal cycler (Applied Biosystems, Taipei, Taiwan); a total volume of 50 μl of PCR reaction solution containing a DNA template (about 50 ng), 75 mM Tris-HCl (pH 8.5), 20 mM ammonium sulfate, 1.5 mM MgCl ₂ , 0.8 mM deoxyribonucleoside triphosphates (0.2 mM each), 1 M (each) primer, and 1.25 U of Hot Start polymerase (Promega, Madison, WI, USA). The PCR reaction was initial denaturation (94 ° C, 3 mins), 40 cycles of denaturation (denaturation, 94 ° C, 1 min), adhesion (annealing, 59 ° C, 1 min) and extension (extension, 72 ° C, 1.5 Mins) reaction, and finally a 7-minute extension reaction (extension, 72 ° C, 7 mins) (Gurtler et al. , 2006), respectively, after amplifying the product, with 2% agar (agarose, , OH, USA) for electrophoresis product analysis. If the PCR product is to be heterozygously reacted with the wafer, each primer is labeled with digoxigenin at one of the [(sp1, sp2) and (Gb1f, Gb1r)] primers or the 5' end of the second primer.

Design of specific probes

The reference strain was sequenced, and the rRNA gene spacer and the gyrB gene sequence of the mycobacteria were collected on the GenBank database, and the sequence alignment (sequence) was performed using the sequence vector software NTI (Invitrogen Corporation, Carlsbad, CA, USA). After alignment, the differential sequences between species and species, as well as sequences of similarities between different species, were designed to design probes. The wafers in this study consisted of 32 probes, including one mycobacteria positive control (code-named PC) and 13 place marker probes (code M) and 31 species-specific probes. Sex probe (Figure 1). The positive control group was selected using the highly conserved region of the mycobacterial rRNA gene spacer collected as much as possible; the coordinate-labeled probe was selected from the labeled digoxigenin-free oligonucleotide-specific oligonucleotide. Probe.

Generation of mock PCR products by means of site direct mutation

In MTBC, the extremely rare species of M. canettii is not available in the center of the world. Therefore, the sequence of the artificially synthesized gyrB gene of Mycobacterium tuberculosis can be used to test whether the probe can identify the tuberculosis. Bacillus. From the MTBC strain collected by GenBank, it was found that there was only one nucleotide difference (SNP) between the gyrB gene sequences of Mycobacterium tuberculosis and Mycobacterium tuberculosis, so the gyrB fragment of Mycobacterium tuberculosis H37Rv was selected and reused. II site-directed mutagenesis kit (Stratagene Products, Hwy, USA), which can produce a gyrB PCR product of Mycobacterium tuberculosis by making a single nucleotide difference in the gyrB sequence of Mycobacterium tuberculosis H37Rv strain. After confirming the correctness of the sequence, the bacterial plastids were taken with a set (Geneaid plasmid midi kit, Taiwan), and the simulated fragments were collected and stored at -80 °C for later use. Since the similarity of the rRNA gene spacer sequences of all the strains in the MTBC was 100%, in the test of Mycobacterium tuberculosis, the hybridization reaction was carried out by PCR products of the rRNA gene spacers of the simulated Mycobacterium tuberculosis gyrB and Mycobacterium tuberculosis H37Rv.

Minute Mycobacterium wafer preparation

The probe and the tracking dye were mixed in a 96-well round bottom microplate (ELISA plate, Becton Drive, NJ, USA) at a volume ratio of 1:1 (vol/vol), and the final concentration of the probe was divided by positive and negative. The group and the coordinate probe were all 10 μM; the positive control (PC) concentration was 5 μM, the coordinate probe concentration was 2.5 μM, and the negative control (NC) was from sterile water and The dye is mixed in equal volume.

The configuration of the microplate was placed Ezspot ^TM arrayer SR-A300 (Wyatt Health Technology, Taipei, Taiwan), using a needle having a diameter of 400 μm of solid (solid pin) points in the respective probe nylon membrane (positively charged Nylon membrane, On Roche, Mannheim, Germany, the center distance between the two points is 800 μm. There are 49 points (7 × 7 dots) on the wafer and the size is 6.8 mm × 6.8 mm. After the wafer was fabricated, it was irradiated with ultraviolet light of 3 W/cm ^{2 of} shortwave UV (Stratalinker 1800; Stratagen, La Jolla, CA, USA) for 30 seconds to promote the probe on the nylon membrane, and then stored in a dark dry place. . The microarray wafer probe locations are shown in Figure 1.

Hybridization of mycobacterial wafers (chip hybridization)

The PCR product of the labeled digoxigenin was separately added to the prepared mycobacterial wafer to carry out a heterozygous reaction. The main steps of the heterozygous reaction are as follows:

1. After labeling the prepared wafer, use 0.5× SSC buffer [1×SSC buffer for 0.15 M NaCl and 0.015 M sodium citrate)-0.1% sodium dodecyl sulfate (SDS, Boehringer Mannheim, Mannheim, Germany)] The wafer was cleaned and shaken 4 times for 3 minutes each time to wash away trace dye on the wafer, probes not bonded to the nylon membrane, and other impurities.

2. Add hybridization solution [hybridization solution, 5 × SSC, 1% (w / v) blocking reagent (Roche Diagnostics, Indianapolis, IN, USA), 0.1% N-laurylsarcosine and 0.02% SDS], at room temperature Prehybridization before shaking for 2 hours

3. Place each wafer sequentially into each well of a 24-well cell culture plate, adding 300 μl of hybrid buffer to each well.

4. Heat the PCR product with digoxigenin to 95 ° C for 5 minutes to denature into a single strand and immediately place on ice.

5. On the ice bath, 10 μl of each single-stranded PCR product was added to the wafer containing the hybrid buffer in a hybridization oven (Firstek Scientific, Taipei, Taiwan) (50 ° C, Hybrid reaction at 1.5 rpm for 1.5 hours

6. Wash the unbound PCR product and hybrid buffer with 0.25 x SSC buffer as wash buffer and wash 4 times for 5 minutes each time.

7. Remove the washing buffer, add 1× stuffing buffer [blocking buffer, 10% blocking reagent (Roche Diagnostics) diluted 10 times with maleic acid buffer (0.1 M maleic acid, 0.15 M NaCl, pH 7.5)], room temperature Shock for 1 hour

8. Add 2500 times diluted (diluted with 1×blocking buffer) with anti-digoxigenin-AP Fab fragments (Roche) labeled with alkaline phosphatase (AP), room temperature shaking reaction 1 hour

9. Remove the antibody buffer, then wash the unbound antibody with buffer [0.3% (v/v) Tween 20 in 1 × maleic acid buffer] and wash 3 times at room temperature for 15 minutes each time.

10. Add detection buffer (0.1 M Tris-HCl, 0.15 M NaCl, pH 9.5) for 5 minutes at room temperature, remove the detection buffer.

11. Dilute the alkaline phosphatase substrate (stock solution of nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolylphosphate (NBT/BCIP), Roche) 50 times with the detection buffer. 40 μl, protected from light at 37 ° C, for a color reaction from 25 minutes to 45 minutes, do not shake during the reaction.

12. Wash the wafer 4 times with pure water for 5 minutes each time to wash off NBT/BCIP to terminate the color reaction.

13. After drying in an oven, you can read the results with the naked eye.

Interpretation of the results of wafer heterozygous reaction

The wafer has coordinate marks on the top and bottom, and the coordinate marks divide the wafer into two large areas. Probes in the upper half of the wafer were used to identify MTBC and its different strains, and two negative control (NC); probes in the lower half were used to identify NTM, and a positive control group ( A positive control, PC) probe that reacts with mycobacteria.

The result of the wafer reaction must have no heterozygous reaction at the NC point, and the PC point has a heterozygous reaction to perform the result interpretation. If both probes for the identification of MTBC are reactive, it indicates that MTBC is present in the sample. As for the MTBC, it will be described in detail below. The identification of NTM is based on the heterozygous reaction of the NTM probe.

rRNA gene spacer, 16S rRNA gene and gyrB sequencing

If the species name of the wafer does not match the species name of the center of the strain, or if it is inconsistent with the name of the species identified by the traditional (or other meristem method), the rRNA gene spacer, 16S rRNA gene, or The gyrB sequence serves as a third reference method. The purified PCR products were sequenced and sequenced using a nucleic acid sequencer PRISM 3100 Sequencer (Applied Biosystems, Taipei, Taiwan), and a BigDye Terminator cycle sequencing kit (version 3.7; Applied Biosystems).

Sensitivity, specificity, and definition of detection limits

Sensitivity: the percentage of the target strain to be identified that was correctly identified [sensitivity=(no. of target strains correctly identified/total no. of target strains tested) × 100%]

Specificity: the percentage of non-target strains that have not been identified [specificity=(no. of nontarget strains not identified/total no. of nontarget strains tested) × 100%]

Detection limit: The minimum amount of genomic DNA that the strain to be tested can be detected by the wafer.

Mycobacterium spp. positive control (PC) probe

This probe is a Mycobacterium spp. probe designed by the common sequence in the rRNA gene spacer sequence of Mycobacterium spp. The probes are Myc2 and Myc3, since the probe Myc2 cannot cover all Mycobacteria, Myc3 was designed for the species that could not be covered, and then the two were mixed together as a probe of mycobacteria, codenamed PC. In this study, when the probe is heterozygous, it indicates that the strain to be tested is a Mycobacterium.

Identification of Mycobacterium tuberculosis (MTBC)

The specific probes of the Mycobacterium tuberculosis group are divided into two parts, one is a flora-specific probe designed for MTBC, and the other is a strain probe designed for each strain in MTBC.

There are two probes in the MTBC flora, codenamed MTBC2 and MTBC4, respectively. The length of the probe is 18 and 19 nucleotides, respectively. The sequence is shared by the sequence shared by the rRNA gene spacer of MTBC. There are great differences; all strains belonging to the Mycobacterium tuberculosis group, including Mycobacterium tuberculosis, Mycobacterium tuberculosis, Mycobacterium tuberculosis, Mycobacterium tuberculosis, Mycobacterium tuberculosis, and Mycobacterium tuberculosis Hybrid reaction; no species other than Mycobacterium tuberculosis will react with the two probes.

There are 12 specific probes for MTBC strains. The probes Mtub1R and MtubW1R are used to identify Mycobacterium tuberculosis. If the strain is Mycobacterium tuberculosis, the hybrid signal of Mtub1R will be stronger than MtubW1R. MbovG1 and MbovGW1 are used to identify Mycobacterium tuberculosis. The heterozygous signal ratio of Mycobacterium tuberculosis, Mycobacterium tuberculosis BCG, and Mycobacterium tuberculosis BCG and MbovG1 probes is stronger than that of MbovGW1, but the strains in the Mycobacterium tuberculosis group cannot be distinguished. Mbov1 and MbovW1 were used to identify Mycobacterium tuberculosis, including Mycobacterium tuberculosis and Mycobacterium tuberculosis BCG and Mbov1. The hybrid signal was stronger than the MbovW1 hybrid signal; Mcap1 and McapW1 were probes for Mycobacterium tuberculosis, only Kapo Mycobacterium tuberculosis will show a strong hybrid signal of Mcap1 than McapW1; Mmic1 and MmicW1 are probes of Mycobacterium tuberculosis, only M. tuberculosis will show a strong hybrid signal of Mmic1 than MmicW1; Mcan1 and McanW1 are the probes of Mycobacterium tuberculosis Because the sequence of the Mycobacterium tuberculosis and Mycobacterium tuberculosis are similar in the gyrB gene, the heterozygous reaction of M. tuberculosis will show a strong hybrid signal of Mtub1R than MtubW1R, and a stronger reaction signal of Mcan1 than McanW1.

The identification of MTBC strains first requires the microbial probes MTBC2 and MTBC4 to react at the same time (ie, the tested species belong to MTBC), and then further identify the species in the group, in a heterozygous pattern ( Hybridization pattern) is used as the basis for identification of strains. Take Mycobacterium tuberculosis as an example, its pattern type is: MTBC2 and MTBC4 react at the same time, Mtub1R probe is stronger than MtubW1R, and there are 10 probes for identifying strains in MTBC, which are also proved to be not in MTBC. Other strains (ie probes with the "W" letter in the probe design have a stronger reaction than probes with the "W" code in the same set). Tuberculosis bacillus was previously placed in the group of Mycobacterium tuberculosis and is now named K. tuberculosis (Aranaz et al., 2003, J. Syst. Evol. Microbiol. 53: 1785-1789), its probe reaction map MbovG1 is more reactive than MbovGW1 (ie belongs to the population of Mycobacterium bovis), and Mcap1 is more reactive than McapW1 (ie it is K. tuberculosis).

Identification of non-tuberculous mycobacteria (NTM)

There are 17 species of probes designed for non-tuberculous mycobacteria. Two of them are complex probes. A total of 19 NTM target strains can be identified, respectively, which are Mycobacterium ulcerans. Mycobacterium avium, Mycobacterium avium, Mycobacterium fortuitum, Mycobacterium tuberculosis, Mycobacterium gordonii, Mycobacterium intracellulare, Mycobacterium kansii, Mycobacterium marinum, Mycobacterium marinum, non-chromosome Mycobacteria, Mycobacterium phlei, Mycobacterium phlei, Mycobacterium phlei, Mycobacterium smegmatis, Mycobacterium smegmatis, Mycobacterium phlei, Mycobacterium ulcerans, and Mycobacterium phlei, among which Mma/ul2 was used to identify Mycobacterium marinum or Mycobacterium ulcerans, and Msi/le1 was used to identify Mycobacterium phlei or Mycobacterium lanatus.

The result of the heterozygous reaction must be positive in the control group (PC), and then the probe is reacted. For example, two probes of PC and Mche3 are reacted, and the strain is identified as Mycobacterium marinum. The only exception is that M. intracellulare has a slight cross-reactivity with the probe for the identification of M. avium (Mavi1), so when Mint2 (a probe for identifying M. intracellulare) and Mavi1 react simultaneously, the strain is identified as a cell. Mycobacterium, but the reaction of Mint2 is much stronger than Mavi1. If PC and Mavi1 have a heterozygous reaction, they are identified as Mycobacterium avium. This is because Mycobacterium avium and Mycobacterium avium belong to Mycobacterium avium complex, both in biochemical reaction and rRNA gene. The spacer sequences are very similar in sequence. It is difficult to distinguish the two strains clinically, and only the Mycobacterium avium group is usually identified.

Wafer test of reference strain

The test results of the wafer are shown in Fig. 2 and Fig. 3. A total of 88 reference strains and one gyrB simulation sequence, including MTBC 15 strain and NTM 73 strain, were tested. The target clinical strains were also tested, including 80 MTBCs (Table 2) and NTM 121 strains (201) (Table 3).

Sensitivity and specificity of mycobacterial wafers

All of the target strains were 289, and two of them ( M. kansasii KMUH0903-4 and M. kansasii KMUH0903-26) were misidentified, so the wafer sensitivity was 99.3% (287/289) (Table 2 and Table 3). Among the 73 strains of nontarget species tested (Table 4), one of them was identified incorrectly ( M. goodii CCUG 52054 was identified as M. smegmatis ), so the wafer specificity was 98.6% (72/73).

Wafer detection limit

Mycobacterium tuberculosis H37Rv, Mycobacterium tuberculosis CCUG 20993 ^T and Mycobacterium tuberculosis JCM 6387 were used as test strains. The DNA concentration was serially diluted by 10 ng/μl. The detection limit of Mycobacterium tuberculosis H37Rv is 10 fg/μl (ie 50 fg/each test), which is equivalent to the DNA content of 12 bacteria [(an E. coli cell contains 4 fg DNA (Kubitschek and Friedman, 1971)] The detection limit of Mycobacterium tuberculosis CCUG 20993 ^T and Occasional Mycobacterium JCM 6387 was 100 fg/μl (ie 500 fg per test), which corresponds to a DNA content of 120 bacteria.

The above embodiments are merely illustrative of the principles and effects of the invention and are not intended to limit the invention. Therefore, those skilled in the art can make modifications and changes to the above embodiments without departing from the spirit of the invention. The scope of the invention should be as set forth in the appended claims.

Figure 1 shows a schematic representation of the position of a microarray wafer probe of the present invention.

Figure 2 shows the heterozygous reaction diagram of the target bacteria of the Mycobacterium tuberculosis group:

(1) Mycobacterium tuberculosis ATCC 25420

(2) Mycobacterium tuberculosis ATCC 35711

(3) Mycobacterium tuberculosis ATCC 27291

(4) Mycobacterium tuberculosis ATCC 35734

(5) Mycobacterium tuberculosis BCG ATCC 35731

(6) Bovine Mycobacterium tuberculosis BCG ATCC 35735

(7) Mycobacterium tuberculosis BCG ATCC 35737

(8) Mycobacterium tuberculosis ATCC BAA-824

(9) Mycobacterium tuberculosis

(10) Mycobacterium tuberculosis ATCC 19422

(11) Mycobacterium tuberculosis ATCC 35782

(12) Mycobacterium tuberculosis NCTC 8337

(13) Mycobacterium tuberculosis H37Rv

(14) Mycobacterium tuberculosis ATCC 25177

(15) Mycobacterium tuberculosis ATCC 35818

(16) Mycobacterium tuberculosis ATCC 35828

Figure 3 shows the heterozygous reaction diagram of non-tuberculous mycobacteria target bacteria (1-19) and non-target bacteria (20-32).

(1) ulcerative tuberculosis CCUG 20993

(2) Mycobacterium avium BCRC 15441

(3) Mycobacterium tuberculosis CCUG 35749

(4) Occasional Mycobacterium BCRC 15320

(5) Mycobacterium tuberculosis CCUG 29062

(6) Mycobacterium gordon CCUG 21801

(7) Mycobacterium intracellulare CCUG 28005

(8) Kansas Mycobacterium CCUG 27785

(9) Mycobacterium lanatus CCUG 47901

(10) Mycobacterium marinum ATCC 25039

(11) Mycobacterium faecalis CCUG 28009

(12) Mycobacterium phlei CCUG 27976

(13) Mycobacterium tuberculosis CCUG 29045

(14) Mycobacterium tuberculosis CCUG 29114

(15) Mycobacterium smegmatis ATCC 19420

(16) Mycobacterium tuberculosis NCTC 10829

(17) Mycobacterium phlei CCUG 42429

(18) Mycobacterium ulcerans ATCC 25899

(19) Mycobacterium phlei ATCC 19973

(20) Mycobacterium M. agri CCUG 37673

(21) Mycobacterium M. alvei CCUG 37585

(22) Mycobacterium M. arupense CCUG 39146

(23) Mycobacterium M. asiaticum MB 031

(24) Mycobacterium M. chitae CCUG 39504

(25) Mycobacterium M. chlorophenolicum BCRC 13726

(26) Mycobacterium M. confluentis CCUG 37513

(27) Mycobacterium M. diernhoferi BCRC 16395

(28) Mycobacterium M. gadium CCUG 37515

(29) Mycobacterium M. pulveris CCUG 37668

(30) Mycobacterium M. fallax CCUG 37584

(31) Mycobacterium M. flavescens MB 017

(32) Mycobacterium M. genavense MB 691

(no component symbol description)

Claims (23)

A method for identifying Mycobacterium spp., which comprises using a probe of Myc2 (SEQ ID No. 1) and Myc3 (SEQ ID No. 2) or a complementary strand thereof to hybridize with DNA in a sample to be tested The reaction, if at least one heterozygous reaction occurs, the sample to be tested comprises mycobacteria. According to the method of claim 1, which further comprises a hybrid reaction using the probe of MTBC2 (SEQ ID No. 3) and MTBC4 (SEQ ID No. 4) or its complementary strand with the DNA in the sample to be tested, such as The DNA in the sample to be tested simultaneously reacts with the probe of MTBC2 (SEQ ID No. 3) and MTBC4 (SEQ ID No. 4) or its complementary strand, and the sample to be tested contains Mycobacterium tuberculosis complex ). According to the method of claim 1, which further comprises a hybrid reaction using the probe of MTBC2 (SEQ ID No. 3) and MTBC4 (SEQ ID No. 4) or its complementary strand with the DNA in the sample to be tested, such as If the DNA in the sample to be tested does not simultaneously hybridize with the probe of MTBC2 (SEQ ID No. 3) and MTBC4 (SEQ ID No. 4) or its complementary strand, the sample to be tested contains non-tuberculous mycobacteria ( Nontuberculous mycobacteria). According to the method of claim 2, which further comprises using a probe of Mtub1R (SEQ ID No. 15) and MtubW1R (SEQ ID No. 16) or a complementary strand thereof to carry out a heterozygous reaction with the DNA in the sample to be tested, such as The intensity of the heterozygous reaction of the DNA in the sample with Mtub1R (SEQ ID No. 15) or its complementary strand is greater than the intensity of the heterozygous reaction between the DNA in the sample to be tested and MtubW1R (SEQ ID No. 16) or its complementary strand. , the sample to be tested contains M. tuberculosis . According to the method of claim 2, which further comprises using a probe of MbovG1 (SEQ ID No. 6) and MbovGW1 (SEQ ID No. 5) or a complementary strand thereof to carry out a hybrid reaction with the DNA in the sample to be tested, such as The intensity of the heterozygous reaction of the DNA in the sample to be tested with MbovG1 (SEQ ID No. 6) or its complementary strand is greater than the heterozygous reaction of the DNA in the sample to be tested with MbovGW1 (SEQ ID No. 5) or its complementary strand. For the intensity, the sample to be tested contains the M. bovis group. The method according to claim 5, wherein the bovine tuberculosis group comprises M. bovis , M. bovis BCG, and M. caprae . According to the method of claim 5, which further comprises using a probe of Mbov1 (SEQ ID No. 8) and MbovW1 (SEQ ID No. 7) or a complementary strand thereof to carry out a heterozygous reaction with the DNA in the sample to be tested, such as The intensity of the heterozygous reaction of the DNA in the sample to be tested with Mbov1 (SEQ ID No. 8) or its complementary strand is greater than the heterozygous reaction of the DNA in the sample to be tested with MbovW1 (SEQ ID No. 7) or its complementary strand. Intensity, the sample to be tested contains Mycobacterium tuberculosis or Mycobacterium tuberculosis BCG. According to the method of claim 5, which further comprises using a probe of Mcap1 (SEQ ID No. 12) and McapW1 (SEQ ID No. 11) or a complementary strand thereof to carry out a hybrid reaction with the DNA in the sample to be tested, such as The intensity of the heterozygous reaction of the DNA in the sample to be tested with Mcap1 (SEQ ID No. 12) or its complementary strand is greater than the heterozygous reaction of the DNA in the sample to be tested with McapW1 (SEQ ID No. 11) or its complementary strand. Intensity, the sample to be tested contains K. tuberculosis. According to the method of claim 2, which further comprises a hybrid reaction using the probe of Mmic1 (SEQ ID No. 14) and MmicW1 (SEQ ID No. 13) or its complementary strand with the DNA in the sample to be tested, such as The intensity of the heterozygous reaction of the DNA in the sample to be tested with Mmic1 (SEQ ID No. 14) or its complementary strand is greater than the heterozygous reaction of the DNA in the sample to be tested with MmicW1 (SEQ ID No. 13) or its complementary strand. Intensity, the sample to be tested contains M. microti . According to the method of claim 2, which further comprises using a probe of Mcan1 (SEQ ID No. 10) and McanW1 (SEQ ID No. 9) or a complementary strand thereof to carry out a heterozygous reaction with the DNA in the sample to be tested, such as The intensity of the heterozygous reaction of the DNA in the sample to be tested with Mcan1 (SEQ ID No. 10) or its complementary strand is greater than the heterozygous reaction of the DNA in the sample to be tested with McanW1 (SEQ ID No. 9) or its complementary strand. For the intensity, the sample to be tested contains M. canettii . According to the method of claim 2, further comprising performing a hybrid reaction with the DNA in the sample to be tested using the first group probe or its complementary strand and the second group probe or its complementary strand, such as in the sample to be tested The intensity of the heterozygous reaction between the DNA and the second group of probes or their complementary strands is greater than the intensity of the heterozygous reaction between the DNA in the sample to be tested and the first group of probes or their complementary strands, and the sample to be tested contains African tuberculosis M. africanum , wherein the first group of probes comprises Mtub1R (SEQ ID No. 15), MbovG1 (SEQ ID No. 6), Mbov1 (SEQ ID No. 8), Mcap1 (SEQ ID No. 12) Mmic1 (SEQ ID No. 14) and Mcan1 (SEQ ID No. 10); the second group of probes comprises MtubW1R (SEQ ID No. 16), MbovGW1 (SEQ ID No. 5), MbovW1 (SEQ ID No. 7) ), McapW1 (SEQ ID No. 11), MmicW1 (SEQ ID No. 13), and McanW1 (SEQ ID No. 9). According to the method of claim 3, which further comprises using one or more selected from the group consisting of Mabs1 (SEQ ID No. 17), Mavi1 (SEQ ID No. 18), Mche3 (SEQ ID No. 19), Mfor1 (SEQ ID No. 20), Mgas2 (SEQ ID No. 21), Mgor1 (SEQ ID No. 22), Mint2 (SEQ ID No. 23), Mkan1 (SEQ ID No. 24), Msi/le1 (SEQ ID No. 25), Mma/ul2 (SEQ ID No. 26), Mnon1 (SEQ ID No. 27), Mper1 (SEQ ID No. 28), Mscr1 (SEQ ID No. 29), Msme3 (SEQ ID No. 31), Mszu1 (SEQ a probe of ID No. 32), Mter1 (SEQ ID No. 33), Mxen2 (SEQ ID No. 35) and its complementary strand is hybridized with DNA in the sample to be tested; as in the sample to be tested the DNA and Mabs1 (SEQ ID No.17) or a complementary probe shares producing a hybrid reactor, the swollen test sample comprising Mycobacterium ulcer (M.abscessus); if the test sample of DNA with Mavi1 ( SEQ ID No. 18) The probe or its complementary strand produces a hybrid reaction, and the sample to be tested comprises M. avium ; such as DNA in the sample to be tested and Mche3 (SEQ ID No. 19) The probe or its complementary strands produce a heterozygous reaction, and the sample to be tested comprises M. chelonae ; such as the DNA in the sample to be tested and Mfor1 (SEQ I D No. 20) The probe or its complementary strands produce a heterozygous reaction, and the sample to be tested comprises M. fortuitum ; such as the DNA in the sample to be tested and Mgas2 (SEQ ID No. 21) The needle or its complementary strands produce a heterozygous reaction, and the sample to be tested comprises M. gastri ; if the DNA in the sample to be tested and the Mgor1 (SEQ ID No. 22) probe or its complementary strand are produced In the hybrid reaction, the sample to be tested comprises M. gordonae ; if the DNA in the sample to be tested is heterozygous with the Mint2 (SEQ ID No. 23) probe or its complementary strand, The sample to be tested comprises M. intracellulare ; if the DNA in the sample to be tested is heterozygous with the Mkan1 (SEQ ID No. 24) probe or its complementary strand, the sample to be tested comprises a bacterium of M. kansasii ; if the DNA in the sample to be tested is heterozygous with the Msi/le1 (SEQ ID No. 25) probe or its complementary strand, the sample to be tested comprises Mycobacterium sputum (M.simiae) or blue M. (M.lentiflavum); if the test sample of DNA with Mma / ul2 (SEQ ID No.26) or a complementary probe shares reaction producing a hybrid, be the Sample contains Mycobacterium marinum (M.marinum) or M. ulcerans (M.ulcerans); if the test sample of DNA with Mnon1 (SEQ ID No.27) or a complementary probe shares that a hybrid reactor, The sample to be tested comprises M. nonchromogenicum ; if the DNA in the sample to be tested is heterozygous with the Mper1 (SEQ ID No. 28) probe or its complementary strand, the test is to be tested. The sample contains M. peregrinum ; if the DNA in the sample to be tested is heterozygous with the Mscr1 (SEQ ID No. 29) probe or its complementary strand, the sample to be tested contains the sputum M. scrofulaceum ; if the DNA in the sample to be tested is heterozygous with the Msme3 (SEQ ID No. 31) probe or its complementary strand, the sample to be tested comprises Mycobacterium smegmatis ( M. Smegmatis ); if the DNA in the sample to be tested is heterozygous with the Mszu1 (SEQ ID No. 32) probe or its complementary strand, the sample to be tested comprises M. szulgai ; and the test sample DNA Mter1 (SEQ ID No.33) or a complementary probe shares producing a hybrid reactor, the test sample comprises M. terrae (M.terrae); As the test sample DNA with Mxen2 (SEQ ID No.35) or a complementary probe shares producing a hybrid reactor, the test sample contains Mycobacterium toad (M.xenopi). The method of any one of claims 1 to 12, wherein the sample to be tested comprises a DNA fragment amplified by a polymerase chain reaction. According to the method of claim 13, it is amplified using sp1 (SEQ ID NO. 36) and sp2 (SEQ ID NO. 37) primers or using Gb1f (SEQ ID NO. 38) and Gb1r (SEQ ID NO. 39) primers. . The method of claim 14, wherein the primer comprises a flag. According to the method of claim 15, wherein the marker is digoxigenin. The method of any one of claims 1 to 12, wherein the hybridization reaction is carried out on a microarray. A probe set comprising Myc2 (SEQ ID No. 1) and Myc3 (SEQ ID No. 2) or a complementary strand thereof. The probe set according to claim 18, further comprising a probe selected from the group consisting of MTBC2 (SEQ ID No. 3), MTBC4 (SEQ ID No. 4), MbovG1 (SEQ ID No. 6), MbovGW1 (SEQ ID No. 5), Mbov1 (SEQ ID No. 8), MbovW1 (SEQ ID No. 7), Mcan1 (SEQ ID No. 10), McanW1 (SEQ ID No. 9) Mcap1 (SEQ ID No. 12), McapW1 (SEQ ID No. 11), Mmic1 (SEQ ID No. 14), MmicW1 (SEQ ID No. 13), Mtub1R (SEQ ID No. 15), MtubW1R (SEQ ID) No. 16), Mabs1 (SEQ ID No. 17), Mavi1 (SEQ ID No. 18), Mche3 (SEQ ID No. 19), Mfor1 (SEQ ID No. 20), Mgas2 (SEQ ID No. 21), Mgor1 (SEQ ID No. 22), Mint2 (SEQ ID No. 23), Mkan1 (SEQ ID No. 24), Msi/le1 (SEQ ID No. 25), Mma/ul2 (SEQ ID No. 26), Mnon1 (SEQ ID No. 27), Mper1 (SEQ ID No. 28), Mscr1 (SEQ ID No. 29), Msme3 (SEQ ID No. 31), Mszu1 (SEQ ID No. 32), Mter1 (SEQ ID No.) 33) and Mxen2 (SEQ ID No. 35) and its complementary strands. A kit for identifying a mycobacterium in a sample, the kit comprising the probe set according to claim 18 or 19. According to the kit of claim 20, wherein the probe is applied to a microarray wafer. According to the set of claim 20, which further comprises a polymerase chain reaction Reagents should be used. According to the kit of claim 22, it comprises sp1 (SEQ ID NO. 36) and sp2 (SEQ ID NO. 37) primers and/or Gb1f (SEQ ID NO. 38) and Gb1r (SEQ ID NO. 39) primers.