A primer, probe composition for nontuberculous mycobacterium typing and application thereof

Abstract

The application discloses a primer, a probe composition for nontuberculous mycobacteria typing and application thereof, and provides a PCR primer and a probe for rapid detection and typing of 13 common NTM in clinic based on genome big data for carrying out pan-genomics analysis, and the specific genes of the common NTM in clinic are screened by using a pan-genomics method, and then typing primers and probes are designed based on the genes, so that the problems of insufficient accuracy and specificity in NTM typing are solved; the various NTM specific sequences screened guarantee the accuracy and specificity; the high sensitivity of the PCR method and the specificity of the sequences guarantee that various interference substances in the clinical samples can be avoided, and the clinical samples can be directly analyzed; the PCR detection is low in cost and low in equipment requirement, and can be carried out in primary hospitals, thereby providing an efficient and economical solution for the precise diagnosis of nontuberculous mycobacteria, the control of disease prevalence and the large-scale disease screening.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of non-tuberculous mycobacterial nucleic acid detection, in particular to a primer, a probe composition for non-tuberculous mycobacterial typing and application thereof. BACKGROUND

[0002] Non-tuberculous mycobacteria (NTM) refers to mycobacteria other than the Mycobacterium tuberculosis complex and Mycobacterium leprae. There are more than 190 known NTMs, about one-third of which can infect humans, causing infection of important tissues and organs such as lungs, lymph nodes and joints and causing systemic disseminated diseases, and even death in severe cases. In recent years, the incidence of NTM lung disease has rapidly increased worldwide.

[0003] Studies have shown that the symptoms and imaging manifestations of diseases caused by different NTMs are very similar, but the treatment regimens are quite different. If NTM patients are treated blindly, drug-resistant strains are likely to be produced, and the condition is likely to worsen. In order to better diagnose and treat and prevent and control NTM infection, it is urgent to develop a rapid, accurate and simple method for identifying common NTM species in clinical practice. Molecular diagnostic techniques provide new possibilities for rapid detection and accurate identification of NTM infection and play a key role in early diagnosis.

[0004] The simplest and fastest molecular diagnostic method applied in clinical practice is mainly a typing method based on specific gene sequences, including PCR (Polymerase Chain Reaction) technology, direct hybridization probe method, linear probe technology (LPA), gene chip method, reverse dot blot hybridization method (REBA), multicolor melting curve method, etc. Among the above methods, the PCR method has the characteristics of low detection cost, low equipment requirement, and can be carried out in primary hospitals, and is widely used in the current mycobacterial typing detection. PCR is a molecular biology technique for amplifying specific DNA fragments in vitro. Its basic principle is to use mother strand DNA as a template, through the cycle of denaturation, annealing and extension, to synthesize daughter strand DNA complementary to the mother strand template in vitro by using DNA polymerase. Based on this, asymmetric PCR, qPCR and multiplex PCR techniques have been widely used in gene typing.

[0005] The existing PCR technology for NTM typing has problems of insufficient accuracy and specificity. The NTM bacterial strain PCR primer is usually designed based on a certain or certain specific gene, such as 16S RNA encoding gene (16S DNA), 16S-23S rRNA intergenic region (ITS), RNA polymerase β subunit (rpoB) and heat shock protein 65 (hsp65) encoding gene, and the obtained product has limited difference between each mycobacterium strain, resulting in extensive cross-reactivity between the target NTM bacterial strains. For example, it cannot distinguish between, for example, Mycobacterium chelonae and Mycobacterium abscessus, Mycobacterium kansasii and Mycobacterium gastri, and Mycobacterium marinum and Mycobacterium ulcerans. CN104131100B discloses a fluorescent PCR reaction solution for identifying 12 kinds of mycobacteria, which comprises amplification primers and molecular beacon probes; the molecular beacon probe has a fluorescent group and a quencher group at both ends. The pair of primers and 12 probes provided by the invention specifically detect Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium abscessus, Mycobacterium fortuitum, Mycobacterium kansasii, Mycobacterium chelonae, Mycobacterium gordonae, Mycobacterium crocolum, Mycobacterium smegmatis, Mycobacterium marinum, Mycobacterium scrofulaceum and Mycobacterium phlei, but the invention cannot distinguish between the clinically very common Mycobacterium avium and Mycobacterium intracellulare. However, the Myco-Panel method based on multiplex PCR cannot distinguish between Mycobacterium marinum and Mycobacterium ulcerans (Microbiol Spectr. 2023, 11(3): e0516222). In the inventor's previous research, the multiplex RT-PCR method developed for identifying major pathogenic mycobacteria in clinical samples cannot distinguish between Mycobacterium avium / intracellulare and Mycobacterium chelonae / abscessus (Heliyon. 2024, 11(1): e41384).

[0006] In the prior art, it is not possible to simultaneously distinguish between the above 13 non-tuberculous mycobacteria, including Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium massiliense, Mycobacterium abscessus, Mycobacterium chelonae, Mycobacterium kansasii, Mycobacterium fortuitum, Mycobacterium crocolum, Mycobacterium marinum, Mycobacterium ulcerans, Mycobacterium gordonae, Mycobacterium phlei and Mycobacterium simiae. Therefore, it is of great significance to provide a method that is specific, sensitive, can distinguish between clinically common NTM and has low equipment requirements, and to lay the foundation for the accurate diagnosis and control of NTM in primary hospitals. SUMMARY

[0007] The purpose of the embodiment of the present application is to provide a primer and probe composition for non-tuberculous mycobacterium typing and its application. The composition is used for the typing and identification of 13 non-tuberculous mycobacteria, has high sensitivity and good specificity, and can quickly type NTM from cultures to guide the development of clinical treatment plans.

[0008] The application is based on genome big data for pan-genomics analysis, screening and providing a PCR primer and probe for rapid detection and typing of 13 common non-tuberculous mycobacteria in clinic, which is designed based on strain-specific genes. The application can solve the problems of insufficient accuracy and specificity in the existing PCR technology for detecting non-tuberculous mycobacteria strains. The technical scheme adopted is as follows:

[0009] In a first aspect, the application provides a primer and probe composition for non-tuberculous mycobacterium typing detection, characterized in that it comprises specific primers and probes for 13 non-tuberculous mycobacteria, including avium mycobacterium, intracellular mycobacterium, massi mycobacterium, abscess mycobacterium, turtle mycobacterium, kansas mycobacterium, sporadic mycobacterium, toad mycobacterium, sea mycobacterium, ulcer mycobacterium, gordon mycobacterium, paragordon mycobacterium and monkey mycobacterium.

[0010] The specific primer sequences and probe sequences of the 13 non-tuberculous mycobacteria are shown in SEQ ID NO: 1-39, and the detailed sequence information is shown in Table 2.

[0011] In some embodiments, the primer and probe composition for non-tuberculous mycobacterium typing detection is the reverse complementary sequence of SEQ ID NO: 1-39.

[0012] In some embodiments, the probe sequence is labeled with a fluorescent group at the 5' end and a quencher group at the 3' end.

[0013] In some embodiments, the fluorescent group is selected from any one of FAM, VIC, TET, JOE, HEX, CY3, CY5, TAMRA, ROX, Texas Red, CY5.5 or CY7.

[0014] In some embodiments, the quencher group is selected from any one of BHQ0, BHQ1, BHQ2, BHQ3 or MGB.

[0015] In some embodiments, the fluorescent group is FAM and the quencher group is BHQ1.

[0016] In some embodiments, the concentration ratio of the forward primer and reverse primer of the 13 non-tuberculous mycobacterium specific primers to the probe is 1-5:1-5:1-4.

[0017] In some embodiments, the concentration ratio of the forward primer and reverse primer of the 13 non-tuberculous mycobacterium specific primers to the probe is 1:1:1.

[0018] In a second aspect, the application provides a method for non-tuberculous mycobacterium typing detection, comprising the following steps:

[0019] S1: Extracting bacterial genomic DNA in the sample to be tested by using bacterial genomic DNA extraction reagent, and storing at -20~ -80℃;

[0020] S2: Using the bacterial genomic DNA in the sample to be tested as a template, and respectively performing PCR amplification by using the primer and probe composition for non-tuberculous mycobacterium typing detection.

[0021] S3: Interpreting the PCR result according to the fluorescence signal.

[0022] Further, the extraction reagent of the bacterial genomic DNA in the step S1 is 1x Tris-EDTA (TE) buffer and glass beads.

[0023] In some embodiments, the DNA storage temperature in the step S1 is -80℃.

[0024] Further, the extraction reagent is a DNA kit that can be purchased from a conventional biochemical reagent store.

[0025] Further, the PCR amplification reaction condition in the step S2 includes:

[0026] Stage 1: 35-39℃, 2-3min;

[0027] Stage 2: 94℃-96℃, 30s-60s;

[0028] Stage 3: 94℃-96℃, 4s-10s;

[0029] Stage 4: 55℃-60℃, 30s-60s;

[0030] Wherein, stages 3 and 4 are repeated for 20-50 cycles.

[0031] In some embodiments, the PCR amplification reaction condition in the step S2 includes:

[0032] Stage 1: 37℃, 2min;

[0033] Stage 2: 95℃, 30s;

[0034] Stage 3: 95℃, 10s;

[0035] Stage 4: 60℃, 30s;

[0036] Wherein, stages 3 and 4 are repeated for 40 cycles.

[0037] Further, the interpretation standard in the step S3 is that the FAM signal is detected at the Ct value of 35 and below.

[0038] In a third aspect, the present application provides a kit for nontuberculous mycobacteria typing detection, comprising the primer probe composition according to any one of the first aspect.

[0039] The present application provides the use of the primer probe composition according to any one of the first aspect or the method for nontuberculous mycobacteria typing detection according to the second aspect or the kit for typing detection according to the third aspect in the preparation of a product for nontuberculous mycobacteria typing detection.

[0040] Compared with the prior art, the present application has the following beneficial effects:

[0041] 1、The primer probe combination of the present application has high specificity and sensitivity, and can accurately identify 13 kinds of clinically common nontuberculous mycobacteria: Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium massiliense, Mycobacterium abscessus, Mycobacterium chelonae, Mycobacterium kansasii, Mycobacterium fortuitum, Mycobacterium marinum, Mycobacterium ulcerans, Mycobacterium gordonae, Mycobacterium gordonae, Mycobacterium gordonae and Mycobacterium simiae in whole genome sequencing data and clinical culture.

[0042] 2、The test method used in the present application is single signal TaqMan probe method qPCR, which has low requirements on equipment and simple result interpretation, and can realize typing detection of 13 kinds of clinically common nontuberculous mycobacteria. The scheme can complete the detection process only by using conventional qPCR instrument, and has the advantages of simplicity, rapidness, comprehensiveness and accuracy. BRIEF DESCRIPTION OF DRAWINGS

[0043] Figure 1 Flow chart for obtaining nontuberculous mycobacteria typing primers and probes by pan-genomics.

[0044] Figure 2 The number of strains and the number of core genes of each species are shown. The number in the bracket represents the number of strains of the species.

[0045] Figure 3 Primer probe composition composition and specificity principle schematic diagram.

[0046] Figure 4 PCR amplification curve diagram of specific primers and probes for Mycobacterium abscessus for cross detection of 13 species. DETAILED DESCRIPTION

[0047] Unless otherwise specifically defined, all technical and scientific terms used in the present application are commonly known to those skilled in the art.

[0048] The application will be further described below in connection with the following drawings and examples, which are only some of the embodiments of the application, but the application is not limited to the following examples.

[0049] The following examples facilitate a better understanding of the present application, but do not limit the present application. In the following examples, the experimental methods are conventional methods unless otherwise specified. In the following examples, the experimental materials used are commercially available from conventional biochemical reagent stores unless otherwise specified.

[0050] In the table: "+" is positive; "-" is negative.

[0051] Example 1

[0052] Pan-genome refers to the sum of all coding genes of a species, including core genes common to all strains (Core genes), dispensable genome (Dispensable genes) and strain-specific genome (Strain-specific genes). By performing pan-genomic analysis on all NTM species and subspecies with available genome sequences, we can identify genes in the dispensable genome that are present in all strains of a particular species, i.e. species-specific core genes. The conserved regions on these genes provide potential sources of typing primers and probes.

[0053] According to the background of pan-genomics, the present application studies non-tuberculous mycobacteria by the following steps:

[0054] (1) Mycobacterium genome download and selection

[0055] The published Mycobacterium genomes were downloaded from the Genome database of NCBI, and a total of 10666 spliced chromosome genomes were obtained. Based on the reference genomes of each species provided by NCBI, the fastANI software was used to confirm the genomes of the target species. If the ANI of the downloaded genome and the reference genome is not less than 95%, the strain belongs to the species. Since the ANI of Mycobacterium parafinlandii and Mycobacterium intracellulare is more than 95%, and the literature reports that the genomic diversity between Mycobacterium intracellulare and Mycobacterium parafinlandii still stays at the subspecies or gene variant level, and does not reach the species level (BMC Microbiol.2021, 21(1):103), therefore, Mycobacterium parafinlandii is regarded as Mycobacterium intracellulare in the present application. The ANI of Mycobacterium ulcerans and Mycobacterium marinum is also more than 95%, but since they have different clinical symptoms, they can only be classified into two different species. At the same time, the members of the corresponding Mycobacterium ulcerans group (MUG) were reclassified according to the existing literature, i.e. Mycobacterium ulcerans and other mycospheroid-producing mycobacteria (MPM) such as Mycobacterium abscessus, Mycobacterium massiliense and Mycobacterium seoulense. Mycobacterium ulcerans M. ​pseudoshottsii M. shinshuense M. liflandii and part of M. marinum M. ulcerans, as these strains have highly consistent genomes, a common ancestor (PLoS Negl Trop Dis. 2010, 4(7):e663, BMC Genomics. 2012, 13:258), and are clearly distinct from the gene content of M. marinum strains.

[0056] After the species were determined, 887 genomes with good assembly results were selected for pan-genomic analysis. These genomes involved M. tuberculosis and 155 non-tuberculosis mycobacteria, including 85 strains of M. avium (MAV), 62 strains of M. intracellulare (MINT), 5 strains of M. marinum (MAR), 35 strains of M. chelonae (MCH), 38 strains of M. abscessus (MAB), 16 strains of M. kansasii (MKA), 4 strains of M. gordonae (MGO), 5 strains of M. paragordonae (MPGO), 26 strains of M. fortuitum (MFO), 12 strains of M. marinum (MMA), 6 strains of M. ulcerans (MUL), 3 strains of M. simiae (MSI), and 7 strains of M. xenopi (MXE). M. avium M. intracellulare M. marseillense M. chelonae M. abscessus M. kansasii M. gordonae M. paragordonae M. fortuitum M. marinum M. ulcerans M. simiae M. xenopi

[0057] (2) Screening of candidate target genes by pan-genomic analysis

[0058] The genomes of the above 887 strains were annotated using Prokka software, and pan-genomic analysis was performed using Roray software to obtain the core genes of each target species. Figure 2 The core genes specific to each target species were screened, and the medium-length members thereof were used as candidate target genes (Table 1).

[0059] Table 1

[0060]

[0061] Example 2

[0062] ​​​​​​​​​​​​​​​The Mafft software was used to perform multiple sequence alignment on the candidate target genes to determine the conserved regions within each target gene. The longest possible conserved regions were selected for primer and probe design. The probe should meet the requirements of a length of 20-30 bp, a GC content of 40%-60%, a melting temperature difference of 5-10°C from the PCR primer, and no secondary structure formation. The primer software was used for primer design. The details of the primers and probes obtained by screening 13 groups of non-tuberculous mycobacterial sequences are shown in Table 2.

[0063] Table 2

[0064]

[0065]

[0066] The probe sequence is labeled with a fluorescent group FAM at the 5' end and a quenching group BHQ1 at the 3' end.

[0067] Example 3

[0068] The above downloaded 10666 mycobacterial genomes were subjected to Blast with the above primers and probes, and the reverse complementary sequences of the primers and probes, and the results were screened for 100% sequence identity and length. If a strain has the forward primer, reverse primer and probe of a species, it is considered to belong to the species. The results showed that the specificity of the above primers and probes for target species identification was 100%. The sensitivity for identifying 10 species of birds Mycobacterium, turtle Mycobacterium, Gordon Mycobacterium, intracellular Mycobacterium, Kansas Mycobacterium, horse Mycobacterium, paragordon Mycobacterium, monkey Mycobacterium, ulcerative Mycobacterium and toad Mycobacterium was also 100%. The sensitivity for identifying other species was above 97%, except for marine Mycobacterium (94.4%), and the detailed results are shown in Table 3.

[0069] Table 3

[0070]

[0071] Example 4

[0072] The PCR detection method used in the embodiment is TaqMan probe method, which is a method of fluorescence detection using TaqMan fluorescent probe. The basic principle is that in the PCR amplification system, in addition to specific primers, a TaqMan probe completely complementary to the target sequence is added, the 5' end of which is labeled with a reporter fluorescent group (FAM), and the 3' end is labeled with a quencher fluorescent group (BHQ). The probe is designed in the amplification region of the upstream and downstream primers and specifically binds to the complementary region of the template DNA in the annealing stage, at which time the quencher group inhibits the reporter group from emitting light through fluorescence resonance energy transfer (FRET); after entering the extension stage, the 5'→3' exonuclease activity of Taq DNA polymerase hydrolyzes the probe, separates the reporter group from the quencher group, and releases the fluorescence signal. The number of cut fluorescent molecules is proportional to the amount of PCR product, so detecting the fluorescence intensity in the PCR reaction system can achieve the purpose of detecting the amplification amount of the PCR product.

[0073] The present application designs a forward primer, a reverse primer and a TaqMan probe (TaqMan probe method qPCR) in the conserved region obtained by the above-mentioned pan-genomic analysis, the distance between the forward primer and the reverse primer is in the range of 50-300 bp, and the TaqMan probe is located between the forward primer and the reverse primer. 2+ When the target bacteria to be detected are added to the amplification system (containing dNTP, Taq enzyme, Mg 2+ and related buffer, forward primer, reverse primer and TaqMan probe), the amplification reaction is started, the 5'-3' exonuclease activity of Taq enzyme degrades the probe, and the fluorescence signal is monitored by the system. In this experiment, different forward primers, reverse primers and probes are designed according to different bacteria and their subspecies to detect the accuracy of the designed system. The composition and specificity principle of the primer probe combination are shown in Figure 3 Only when the fluorescence signal and amplification curve appear in the system (containing dNTP, Taq enzyme, Mg 2+ and related buffer, forward primer, reverse primer and TaqMan probe) with the addition of nucleic acid template of target bacteria matched with the designed bacteria, the detection system is designed correctly and has bacteria specificity.

[0074] The mycobacterial DNA in the sputum sample or the cultured bacteria liquid sample of the person to be examined is amplified by PCR, and the amplification primer sequence is one of the 13 pairs of typing primers. At the same time, the corresponding probe is added to the PCR reaction solution.

[0075] In addition to the above-mentioned primers and probes, the PCR reaction solution also includes 5 μL 2× Animal Detection U+Probe qPCR Super PreMix premix, which contains DNA polymerase (Taq enzyme), related buffer, Mg 2+dNTPs, etc. to prepare the PCR reaction solution and to perform PCR amplification. The amplification procedure can include denaturation, annealing, and extension.

[0076] The binding of the probes to the PCR products and the reaction procedure are as follows: after each probe binds to the PCR product of the corresponding bacterial species at a low temperature, the 5'-3' exonuclease activity of the Taq enzyme degrades the probe by enzymatic cleavage, separates the reporter fluorescent group and the quencher fluorescent group, and emits fluorescence. The number of cleaved fluorescent molecules is proportional to the number of PCR products. Finally, it is determined whether the sample belongs to the NTM bacterial species based on whether a fluorescent signal is emitted.

[0077] For example, the culture of 13 bacterial species is cross-detected by using the Mycobacterium abscessus primer and probe:

[0078] (a) Extraction of Mycobacterium DNA

[0079] The specimen is selected for pretreatment, such as liquefaction of sputum and colony picking. Then, the Mycobacterium DNA is extracted by using the CapitalBio Mycobacterium RT-PCR kit (CapitalBio RT-PCR, CapitalBio Technology Inc., Beijing, China), and the specific steps are performed according to the kit instructions.

[0080] (b) PCR amplification

[0081] After the PCR reaction solution is prepared, PCR amplification is performed by using the Mycobacterium abscessus, the Mycobacterium maculare standard strain, and the other 11 bacterial species clinically verified by whole genome sequencing, to test the detection accuracy and specificity of the target NTM.

[0082] The PCR amplification reaction conditions include:

[0083] Stage 1: 37°C-2min;

[0084] Stage 2: 95°C-30s;

[0085] Stage 3: 95°C-10s;

[0086] Stage 4: 60°C-30s;

[0087] Among them, stages 3 and 4 are repeated for 40 cycles.

[0088] (c) Result interpretation

[0089] The fluorescence signal of the FAM channel was observed, and when the Ct value was ≤ 35 and a standard S curve was shown, it was judged to be positive; when the Ct value was > 35 or no signal was shown, it was judged to be negative. When the mycobacterium abscessus primer and probe were used to cross-detect 13 strains, only the mycobacterium abscessus showed the FAM fluorescence signal, and the other strains showed no signal Figure 4 ).

[0090] Based on the above steps, the sensitivity and specificity of the 13 NTM typing primers and probes were evaluated, and the results are shown in Table 4. Except for the primers and probes of Mycobacterium chelonae and Mycobacterium ulcerans which had cross-reactions, the primers and probes of the other strains had good sensitivity and specificity. Although the primers and probes of Mycobacterium chelonae could detect MCH and MAB, the primers and probes of MAB had good specificity and could only detect MAB, so MCH could be identified by exclusion. Similarly, the primers and probes of MUL could detect MUL and MMA, but the primers and probes of Mycobacterium marinum could only detect MMA, so MUL could also be identified by exclusion.

[0091] Table 4

[0092]

[0093] Comparative Example 1

[0094] The present application also conducted comparative experiments on 20 common strains other than the 13 NTM, and the specific detection steps were the same as in Example 4. All the primers and probes were negative when detecting other NTM, Mycobacterium tuberculosis, BCG, and common pathogenic bacteria causing pneumonia. This shows that the primers and probe compositions provided by the present application for non-tuberculous mycobacterium typing have good sensitivity and specificity for the target typing strains.

[0095] Table 5

[0096]

[0097] The abbreviations and Latin names of the 20 common strains are as follows: MPE: M. peregrinum (Extracomplesomyces); MTR: M. triplex (Triplex mycobacterium); MLE: M. lentiflavum (M. lentiflavum); MBR: M. brisbanense (Brisbane mycobacterium); MIR: M. iranicum (Iranian mycobacterium); MMAG: M. mageritense (Margaret mycobacterium); MMUC: M. mucogenicum (Mucoid mycobacterium); MNA: M. neoaurum(Mycobacterium neoaurum); MNC: M. novocastrense (Mycobacterium obuense); MOB: M. obuense (Mycobacterium obuense); MSE: M. seoulense (Mycobacterium shimoidei); MSHI: M. shimoidei (Mycobacterium shimoidei); MTB: M. tuberculosis (Mycobacterium tuberculosis); BCG: M. bovis BCG (Bacillus Calmette-Guerin); KP: Klebsiella pneumoniae (Klebsiella pneumoniae); S. aureus : Staphylococcus aureus (Staphylococcus aureus); PA: Pseudomonas aeruginosa (Pseudomonas aeruginosa); E. coli : Escherichia coli (Escherichia coli); C. neoformans : Cryptoccus neoformans (Cryptococcus neoformans); A. fumigatus : Aspergillus fumigatus (Aspergillus fumigatus).

[0098] The present application is not limited to the specific embodiments disclosed and described above. Modifications and variations of the application should fall within the scope of the claims appended hereto and are intended to be covered by the appended claims. Further, although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.

Claims

1. A primer probe combination kit for detecting 13 kinds of clinically common non-tuberculous mycobacteria, characterized in that, comprises a forward primer of the nucleotide sequence set forth in SEQ ID NO: 1, a reverse primer of the nucleotide sequence set forth in SEQ ID NO: 2, and a probe of the nucleotide sequence set forth in SEQ ID NO: 3; (b) a composition for detecting M. intracellulare, comprising a forward primer of the nucleotide sequence set forth in SEQ ID NO: 4, a reverse primer of the nucleotide sequence set forth in SEQ ID NO: 5, and a probe of the nucleotide sequence set forth in SEQ ID NO: 6; (c) a composition for detecting M. avium, comprising a forward primer of the nucleotide sequence set forth in SEQ ID NO: 7, a reverse primer of the nucleotide sequence set forth in SEQ ID NO: 8, and a probe of the nucleotide sequence set forth in SEQ ID NO: 9; (d) a composition for detecting M. massiliense, comprising a forward primer of the nucleotide sequence set forth in SEQ ID NO: 10, a reverse primer of the nucleotide sequence set forth in SEQ ID NO: 11, and a probe of the nucleotide sequence set forth in SEQ ID NO: 12; (e) a composition for detecting M. chelonae, comprising a forward primer of the nucleotide sequence set forth in SEQ ID NO: 13, a reverse primer of the nucleotide sequence set forth in SEQ ID NO: 14, and a probe of the nucleotide sequence set forth in SEQ ID NO: 15; (f) a composition for detecting M. fortuitum, comprising a forward primer of the nucleotide sequence set forth in SEQ ID NO: 16, a reverse primer of the nucleotide sequence set forth in SEQ ID NO: 17, and a probe of the nucleotide sequence set forth in SEQ ID NO: 18; (g) a composition for detecting M. kansasii, comprising a forward primer of the nucleotide sequence set forth in SEQ ID NO: 19, a reverse primer of the nucleotide sequence set forth in SEQ ID NO: 20, and a probe of the nucleotide sequence set forth in SEQ ID NO: 21; (h) a composition for detecting M. marinum, comprising a forward primer of the nucleotide sequence set forth in SEQ ID NO: 22, a reverse primer of the nucleotide sequence set forth in SEQ ID NO: 23, and a probe of the nucleotide sequence set forth in SEQ ID NO: 24; (i) a composition for detecting M. ulcerans, comprising a forward primer of the nucleotide sequence set forth in SEQ ID NO: 25, a reverse primer of the nucleotide sequence set forth in SEQ ID NO: 26, and a probe of the nucleotide sequence set forth in SEQ ID NO: 27; (j) a composition for detecting M. amphibii, comprising a forward primer of the nucleotide sequence set forth in SEQ ID NO: 28, a reverse primer of the nucleotide sequence set forth in SEQ ID NO: 29, and a probe of the nucleotide sequence set forth in SEQ ID NO: 30; (k) a composition for detecting M. gordonae, comprising a forward primer of the nucleotide sequence set forth in SEQ ID NO: 31, a reverse primer of the nucleotide sequence set forth in SEQ ID NO: 32, and a probe of the nucleotide sequence set forth in SEQ ID NO: 33; ​ (l) a composition for detecting M. paragordonae, comprising a forward primer with a nucleotide sequence as set forth in SEQ ID NO: 34, a reverse primer with a nucleotide sequence as set forth in SEQ ID NO: 35, and a probe with a nucleotide sequence as set forth in SEQ ID NO: 36; (m) a composition for detecting M. macacae, comprising a forward primer with a nucleotide sequence as set forth in SEQ ID NO: 37, a reverse primer with a nucleotide sequence as set forth in SEQ ID NO: 38, and a probe with a nucleotide sequence as set forth in SEQ ID NO:

39.

2. The primer probe combination kit for detecting 13 kinds of clinically common non-tuberculous mycobacteria according to claim 1, characterized in that, The sequence is the reverse complement sequence of SEQ ID NO: 1-39. 3.The primer-probe combination kit for detecting 13 clinically common non-tuberculous mycobacteria according to claim 1, characterized in that, The probe is labeled with a fluorescent group at the 5' end and a quencher group at the 3' end. 4.The primer-probe combination kit for detecting 13 kinds of clinically common non-tuberculous mycobacteria according to claim 3, characterized in that, The fluorescent group is selected from any one of FAM, VIC, TET, JOE, HEX, CY3, CY5, TAMRA, ROX, Texas Red, CY5.5 or CY7; and the quencher group is selected from any one of BHQ0, BHQ1, BHQ2, BHQ3 or MGB.

5. The primer-probe combination kit for detecting 13 clinically common non-tuberculous mycobacteria according to claim 1, characterized in that, The concentration ratio of the specific forward primer, reverse primer and probe for the 13 non-tuberculous mycobacteria is 1-5: 1-5: 1-4.

6. Use of the primer-probe combination kit for detecting 13 clinically common non-tuberculous mycobacteria according to any one of claims 1-5 in the preparation of a product for detecting the typing of non-tuberculous mycobacteria.

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