Primer and probe composition for typing nontuberculous mycobacteria and application of primer and probe composition

Abstract

The invention discloses a primer and probe composition for typing nontuberculous mycobacteria and application of the primer and probe composition, and provides a PCR primer and probe for rapid detection and typing of 13 clinical common NTMs based on generic big data, specific genes of clinical common NTM strains are screened by using a generic genomics method, and the specific genes of the clinical common NTM strains are screened by using a PCR detection method. Then typing primers and probes are designed on the basis of the genes, so that the problems of insufficient accuracy and specificity in NTM typing are solved; the accuracy and the specificity are ensured by the screened specific sequences of the NTM; the two factors of high sensitivity and sequence specificity of the PCR method ensure that various interfering substances in a clinical sample can be avoided, and the clinical sample is directly analyzed; the PCR detection cost is low, the equipment requirement is low, and an efficient and economical solution can be provided for precise diagnosis, disease prevalence control and large-scale disease screening of the nontuberculous mycobacteria in primary hospitals.

Description

Technical Field

[0001] The present invention relates to the technical field of non-tuberculous mycobacteria nucleic acid detection, and in particular to a primer and probe composition for non-tuberculous mycobacteria typing and applications thereof. Background Art

[0002] Non-tuberculous mycobacteria (NTM) refer to mycobacteria other than the Mycobacterium tuberculosis complex and Mycobacterium leprae. Currently, there are over 190 known NTM species, approximately one-third of which can infect humans, causing infection of vital tissues and organs such as the lungs, lymph nodes, and joints, leading to systemic disseminated disease and, in severe cases, even death. In recent years, the incidence of NTM lung disease has rapidly increased worldwide.

[0003] Studies have shown that the symptoms and imaging manifestations of different NTM infections are very similar, but treatment options vary significantly. Blindly prescribing medication to NTM patients can easily lead to the emergence of drug-resistant strains and worsen their condition. To better diagnose, treat, and prevent NTM infections, there is an urgent need to develop rapid, accurate, and simple clinical identification methods for common NTM species. Molecular diagnostic technology offers new possibilities for rapid detection and accurate identification of NTM infections, playing a key role in early diagnosis.

[0004] The simplest and most rapid molecular diagnostic methods used in clinical practice are primarily based on specific gene sequence typing methods, including PCR (polymerase chain reaction) technology, direct hybridization probes, line probe assays (LPA), gene chips, reverse blot hybridization assays (REBA), and multicolor melting curve analysis. Among these methods, PCR is widely used for mycobacterial typing due to its low cost, minimal equipment requirements, and availability in primary care hospitals. PCR is a molecular biology technique for amplifying specific DNA fragments in vitro. Its basic principle is to use a parent DNA template and, through a three-step cycle of denaturation, annealing, and extension, utilize DNA polymerase to synthesize daughter DNA strands complementary to the parent template in vitro. Derived from this approach, asymmetric PCR, qPCR, and multiplex PCR techniques have been widely used in genotyping.

[0005] Existing PCR technology for NTM typing has problems with accuracy and specificity. PCR primers for NTM species are usually designed based on only one or certain specific genes, such as the 16S RNA coding gene (16S DNA), the 16S-23S rRNA intergenic region (ITS), the β-subunit of RNA polymerase (rpoB), and the heat shock protein 65 (hsp65) coding gene. The obtained products have limited differences between various mycobacterial species, resulting in extensive cross-reactivity between target NTM species. For example, it is impossible to distinguish between Mycobacterium chelonae and Mycobacterium abscessus, Mycobacterium kansasii and Mycobacterium gastricis, and Mycobacterium marinum and Mycobacterium ulcerans. CN104131100B discloses a fluorescent PCR reaction solution for identifying 12 types of mycobacteria, comprising amplification primers and molecular beacon probes; the molecular beacon probes have a fluorescent group and a quenching group at both ends, respectively. The invention provides a pair of primers and 12 probes that specifically detect Mycobacterium tuberculosis, Mycobacterium avium intracellulare, Mycobacterium abscessus, Mycobacterium fortuitum, Mycobacterium kansasii, Mycobacterium chelonae, Mycobacterium gordonii, Mycobacterium toad, Mycobacterium smegmatis, Mycobacterium marinum, Mycobacterium scrofulae, and Mycobacterium phlei. However, the invention cannot distinguish between Mycobacterium avium and Mycobacterium intracellulare, which are very common in clinical practice. However, the Myco-Panel method based on multiplex PCR cannot distinguish Mycobacterium marinum from 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 could not distinguish between Mycobacterium avium / intracellulare and Mycobacterium chelonae / abscessus (Heliyon. 2024, 11(1):e41384).

[0006] Existing technologies are unable to simultaneously distinguish between the 13 non-tuberculous mycobacteria listed above, including Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium massa, Mycobacterium abscessus, Mycobacterium chelonae, Mycobacterium kansasii, Mycobacterium fortuitum, Mycobacterium toadense, Mycobacterium marinum, Mycobacterium ulcerans, Mycobacterium gordonii, Mycobacterium paragordonii, and Mycobacterium simianum. Therefore, providing a method with high specificity, high sensitivity, the ability to distinguish common clinical NTMs, and minimal equipment requirements is of great significance, laying the foundation for accurate NTM diagnosis and epidemic control in primary care hospitals. Summary of the Invention

[0007] The present invention aims to provide a primer and probe combination for typing nontuberculous mycobacteria and its application. This combination is used to type and identify 13 nontuberculous mycobacteria with high sensitivity and good specificity. It can rapidly perform NTM typing in cultures and guide clinical treatment planning.

[0008] This study uses pan-genomic analysis based on genomic big data to screen and provide a set of PCR primers and probes designed based on species-specific genes for the rapid detection and typing of 13 common clinical non-tuberculous mycobacteria. This approach addresses the limitations of existing PCR technology in detecting the presence of non-tuberculous mycobacteria, which suffer from insufficient accuracy and specificity. The technical solution employed is as follows: In a first aspect, the present invention provides a primer and probe combination for typing detection of non-tuberculous mycobacteria, characterized by comprising specific primers and probes for 13 types of non-tuberculous mycobacteria, including Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium massa, Mycobacterium abscessus, Mycobacterium chelonae, Mycobacterium kansasii, Mycobacterium fortuitum, Mycobacterium toadense, Mycobacterium marinum, Mycobacterium ulcerans, Mycobacterium gordonii, Mycobacterium paragordonii, and Mycobacterium simianum. The specific primer sequences and probe sequences for the 13 non-tuberculous mycobacteria are shown in SEQ ID NOs: 1 to 39, and the detailed sequence information is shown in Table 2.

[0009] In some embodiments, the primer and probe combination for non-tuberculous mycobacterium typing detection is the reverse complementary sequence of SEQ ID NOs: 1-39.

[0010] In some embodiments, the probe sequence is labeled with a fluorescent group at the 5' end and a quencher group at the 3' end; 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.

[0011] In some embodiments, the quencher group is selected from any one of BHQ0, BHQ1, BHQ2, BHQ3 or MGB.

[0012] In some embodiments, the fluorescent group is FAM and the quencher group is BHQ1.

[0013] In some embodiments, the concentration ratio of the forward primer and reverse primer of the 13 non-tuberculosis mycobacteria-specific primers to the probe is 1-5:1-5:1-4.

[0014] In some embodiments, the concentration ratio of the forward primer to the reverse primer to the probe of the 13 non-tuberculosis mycobacteria-specific primers is 1:1:1.

[0015] In a second aspect, the present invention provides a method for typing and detecting nontuberculous mycobacteria, comprising the following steps: S1: Use bacterial genomic DNA extraction reagent to extract bacterial genomic DNA from the sample to be tested and store it at -20~-80℃; S2: using the bacterial genomic DNA in the sample to be tested as a template, PCR amplification is performed using the primer and probe combination for non-tuberculous mycobacterium typing detection; S3: Interpret the PCR results based on the fluorescence signal.

[0016] Furthermore, the extraction reagents for bacterial genomic DNA in step S1 are: 1×Tris-EDTA (TE) buffer and glass beads.

[0017] In some embodiments, the DNA storage temperature in step S1 is -80°C.

[0018] Furthermore, the extraction reagent is a DNA kit that can be purchased from a conventional biochemical reagent store.

[0019] Furthermore, the PCR amplification reaction conditions in step S2 include: Stage 1: 35-39°C, 2-3 minutes; Stage 2: 94°C-96°C, 30s-60s; Stage 3: 94°C-96°C, 4s-10s; Stage 4: 55°C-60°C, 30s-60s; Stages 3 and 4 are repeated for 20-50 cycles.

[0020] In some embodiments, the PCR amplification reaction conditions in step S2 include: Stage 1: 37°C, 2 min; Stage 2: 95°C, 30 seconds; Stage 3: 95°C, 10 seconds; Stage 4: 60°C, 30 seconds; Stages 3 and 4 were repeated for 40 cycles.

[0021] Furthermore, the judgment standard in step S3 is: FAM signal is detected at a Ct value of 35 or below.

[0022] In a third aspect, the present invention provides a kit for typing and detecting non-tuberculous mycobacteria, comprising the primer-probe combination as described in any one of the first aspects.

[0023] The present invention provides a primer-probe combination as described in any one of the first aspect, or the method for typing detection of non-tuberculous mycobacteria as described in the second aspect, or the kit for typing detection as described in the third aspect, and use thereof in preparing products for typing detection of non-tuberculous mycobacteria.

[0024] Compared with the prior art, the present invention has the following beneficial effects: 1. The primer-probe combination of the present invention has high specificity and sensitivity, and can accurately identify 13 common clinical non-tuberculous mycobacteria in whole genome sequencing data and clinical cultures: Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium massa, Mycobacterium abscessus, Mycobacterium chelonae, Mycobacterium kansasii, Mycobacterium fortuitum, Mycobacterium toadense, Mycobacterium marinum, Mycobacterium ulcerans, Mycobacterium gordonii, Mycobacterium paragordonii, and Mycobacterium simiae.

[0025] 2. The test method used in this invention is a single-signal TaqMan probe-based qPCR method, which requires minimal equipment and is easy to interpret. It can detect 13 common clinical non-tuberculosis mycobacteria. This method can complete the detection process using only conventional qPCR instruments, offering the advantages of simplicity, speed, comprehensiveness, and accuracy. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Flowchart for obtaining primers and probes for typing nontuberculous mycobacteria through pan-genomics.

[0027] Figure 2 The numbers on each petal indicate the number of core genes of the corresponding strain, and the numbers in brackets indicate the number of strains of that strain.

[0028] Figure 3 This is a schematic diagram illustrating the composition and specificity principle of the primer-probe combination.

[0029] Figure 4 This is the PCR amplification curve of Mycobacterium abscessus-specific primers and probes cross-detecting 13 bacterial species. DETAILED DESCRIPTION

[0030] Unless otherwise specifically defined, all technical and scientific terms used in the present invention are common knowledge to those skilled in the art.

[0031] The present invention will be further described below with reference to the following drawings and examples. The described examples are only a part of the present invention, but the present invention is not limited to the following examples.

[0032] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples were purchased from conventional biochemical reagent stores unless otherwise specified.

[0033] In the table: “+” represents positive; “-” represents negative.

[0034] Example 1 The pan-genome refers to the sum of all coding genes within a species, including core genes shared by all strains, dispensable genes, and strain-specific genes. By performing pan-genomic analysis on all NTM species and subspecies for which genome sequences are available, we can identify genes within the dispensable genes that are common to all strains of a particular species—strain-specific core genes. Conserved regions within these genes provide potential sources of typing primers and probes.

[0035] Based on the background of pan-genomics, the present invention studies non-tuberculosis mycobacteria through the following steps: (1) Downloading and selecting mycobacterial genomes The published mycobacterium genomes were downloaded from the Genome database of NCBI, and a total of 10,666 spliced ​​chromosome genomes were obtained. Based on the reference genomes of each species provided by NCBI, the fastANI software was used to confirm the genome 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 paraintracellularis and Mycobacterium intracellularis exceeds 95%, and the literature reports that the genomic diversity between Mycobacterium intracellularis and Mycobacterium paraintracellularis remains at the subspecies or gene variant level and has not reached the species level (BMC Microbiol. 2021, 21(1):103), the present invention uses Mycobacterium paraintracellularis as intracellular Mycobacterium. The ANI of Mycobacterium ulcerans and Mycobacterium marinum is also above 95%, but because the two have different clinical symptoms, they can only be divided into two different species. At the same time, reference is made to the existing literature on the corresponding Mycobacterium ulcerans group ( Mycobacterium ulcerans The members of the group were reclassified as Mycobacterium ulcerans and other mycolide-producing mycobacteria (MPM), such as M. pseudoshottsii 、 M. shinshuense 、 M. liflandii and part Marine M. , were all classified as Mycobacterium ulcerans because these strains had highly identical genomes, shared a common ancestor (PLoS Negl Trop Dis. 2010, 4(7):e663, BMC Genomics. 2012, 13:258), and had significantly different gene contents from Mycobacterium marinum strains.

[0036] After the bacterial species were identified, 887 genomes with good assembly results were selected for pan-genomic analysis. These genomes involved Mycobacterium tuberculosis and 155 non-tuberculosis mycobacteria, including Mycobacterium avium (MAV, M. avium ) 85 strains, Mycobacterium intracellulare (MINT, Intracellular M. ) 62 strains, Mycobacterium massoniana (MAR, M. marseillense ) 5 strains, Mycobacterium chelonae (MCH, M. chelonae ) 35 strains, Mycobacterium abscessus (MAB, M. abscess ) 38 strains, Mycobacterium kansasii (MKA, M. kansasii ) 16 strains, Mycobacterium gordonii (MGO, M. gordonae ) 4 strains, Mycobacterium paragordonii (MPGO, M. paragordonae ) 5 strains, Mycobacterium fortuitum (MFO, M. fortuitum ) 26 strains, Mycobacterium marinum (MMA, Marine M. ) 12 strains, Mycobacterium ulcerans (MUL, Ulcerative muscle ) 6 strains, Mycobacterium simianum (MSI, M. simiae ) 3 strains and Mycobacterium toadense (MXE, M. xenopi ) 7 strains.

[0037] (2) Pan-genomic analysis to screen candidate target genes The genomes of the above 887 strains were annotated using Prokka software, and then pan-genomic analysis was performed using Roray software to obtain the core genes of each target strain ( Figure 2 ). Specific core genes of each target bacterial species were screened, and members of equal length were selected as candidate target genes (Table 1).

[0038] Table 1

[0039] Example 2 Multiple sequence alignments of candidate target genes were performed using Mafft software to identify conserved regions across bacterial species within each target gene. Primer and probe design was performed by selecting conserved regions as long as possible. Probes should be between 20 and 30 bp in length, have a GC content of 40% to 60%, have a melting temperature that differs by 5 to 10°C from that of PCR primers, and lack the formation of secondary structures. Primers were designed using Primer software. Details of the primers and probes obtained from the 13 sets of nontuberculous mycobacterial sequence screening are shown in Table 2.

[0040] Table 2

[0041]

[0042] The 5' end of the probe sequence is labeled with the fluorescent group FAM, and the 3' end is labeled with the quencher group BHQ1.

[0043] Example 3 The 10,666 downloaded mycobacterial genomes were blasted against the primers and probes described above, as well as their corresponding reverse complements. Results were screened for 100% sequence identity and length. If a strain had the forward primer, reverse primer, and probe for a particular species, it was considered to belong to that species. The results showed that the primers and probes described above had 100% specificity for identifying the target species. The sensitivity for identifying 10 species, including Mycobacterium avium, Mycobacterium chelonae, Mycobacterium gordonii, Mycobacterium intracellulare, Mycobacterium kansasii, Mycobacterium massa, Mycobacterium paragordonii, Mycobacterium simianum, Mycobacterium ulcerans, and Mycobacterium xenopus, was also 100%. With the exception of Mycobacterium marinum (94.4%), the sensitivity for identifying all other species was above 97%. Detailed results are shown in Table 3.

[0044] Table 3

[0045] Example 4 The PCR detection method used in this example is the TaqMan probe method, which uses TaqMan fluorescent probes for fluorescence detection. The basic principle is: in addition to specific primers, a TaqMan probe fully complementary to the target sequence is added to the PCR amplification system. Its 5' end is labeled with a reporter fluorescent group (FAM) and its 3' end is labeled with a quencher fluorescent group (BHQ). The probe is designed within the amplification regions of the upstream and downstream primers. During the annealing phase, it specifically binds to the complementary region of the template DNA. At this time, the quencher inhibits the reporter group's luminescence through fluorescence resonance energy transfer (FRET). During the extension phase, the 5'→3' exonuclease activity of Taq DNA polymerase hydrolyzes the probe, separating the reporter group from the quencher group and releasing a fluorescent signal. The number of fluorescent molecules cleaved is proportional to the amount of PCR product. Therefore, the amount of PCR product amplification can be determined by measuring the fluorescence intensity in the PCR reaction system.

[0046] The present invention designs a front primer, a back primer and a TaqMan probe (TaqMan probe method qPCR) in the conserved region obtained by the above pan-genome analysis. The distance between the front and back primers is within the range of 50-300 bp. The TaqMan probe is located between the front and back primers. When the amplification system (including dNTP, Taq enzyme, Mg 2+ When the target bacterial species is added to the TaqMan probe (and related buffer, forward primer, backward primer, and TaqMan probe), the amplification reaction is initiated. The 5'-3' exonuclease activity of the Taq enzyme cleaves and degrades the probe, and the system monitors the fluorescent signal. This experiment designs different forward primers, backward primers, and probes according to different bacterial species and subspecies to test the accuracy of the designed system. The composition and specificity principle of the primer probe combination are as follows Figure 3As shown, only when the system (including dNTP, Taq enzyme, Mg 2+ When a nucleic acid template of a target bacterial species that matches the designed bacterial species is added to the assay (and related buffer, forward primer, backward primer, and TaqMan probe), a fluorescent signal and an amplification curve appear, and the detection system is correctly designed and has bacterial species specificity.

[0047] Mycobacterium DNA is amplified by PCR from a sputum sample or cultured bacterial liquid sample of the patient to be examined, wherein the amplification primer sequence is one of the 13 pairs of typing primers. At the same time, the corresponding probe is used and added to the PCR reaction solution.

[0048] In addition to the above primers and probes, the PCR reaction solution also includes 5 μL of 2× Animal Detection U+ProbeqPCR Super PreMix premix, which contains DNA polymerase (Taq enzyme), relevant buffer, Mg 2+ , dNTPs, etc., for the preparation of PCR reaction solution and PCR amplification. The amplification process may include denaturation, annealing, and extension.

[0049] The binding and reaction process between probes and PCR products is as follows: At low temperature, each probe binds to the corresponding PCR product. As the temperature gradually increases, the 5'-3' exonuclease activity of the Taq enzyme cleaves the probe, separating the reporter and quencher fluorophores, resulting in fluorescence. The number of fluorescent molecules cleaved is proportional to the amount of PCR product. Ultimately, the presence of a fluorescent signal determines the NTM species to which the sample belongs.

[0050] Take the cross-detection of 13 strains of Mycobacterium abscessus using primers and probes as an example: (a) Mycobacterial DNA extraction Specimens were pretreated, including sputum liquefaction and colony picking. Mycobacterium DNA was then extracted using the CapitalBioMycobacterium RT-PCR kit (CapitalBio RT-PCR, CapitalBio Technology Inc., Beijing, China). Specific steps were performed according to the kit instructions.

[0051] (b) PCR amplification After preparing the PCR reaction solution, PCR amplification was performed using standard strains of Mycobacterium abscessus, Mycobacterium simianum, and clinical strains of 11 other bacterial species verified by whole genome sequencing to test the detection accuracy and specificity of the target NTM.

[0052] PCR amplification reaction conditions include: Stage 1: 37°C for 2 minutes; Stage 2: 95°C-30s; Stage 3: 95°C for 10 seconds; Stage 4: 60℃-30s; Stages 3 and 4 were repeated for 40 cycles.

[0053] (c) Interpretation of results Observe the fluorescence signal of the FAM channel. When the Ct value is ≤35 and a standard S curve is shown, it is judged as positive; when the Ct value is >35 or no signal is shown, it is judged as negative. When the primers and probes for Mycobacterium abscessus are used to cross-detect 13 bacterial species, only Mycobacterium abscessus shows FAM fluorescence signal, and no signal is shown for other bacterial species ( Figure 4 ).

[0054] Based on the above steps, the sensitivity and specificity of the primers and probes for 13 NTM typing species were evaluated, and the results are shown in Table 4. With the exception of the primers and probes for Mycobacterium chelonae and Mycobacterium ulcerans, which showed cross-reactivity, the primers and probes for the other species all showed excellent sensitivity and specificity. While the primers and probes for Mycobacterium chelonae could detect both MCH and MAB, the primers and probes for MAB had excellent specificity and only detected MAB, allowing MCH to be identified by exclusion. Similarly, the primers and probes for MUL could detect both MUL and MMA, but the primers and probes for Mycobacterium marinum only detected MMA, allowing MUL to be identified by exclusion.

[0055] Table 4

[0056] Comparative Example 1 The present invention also conducted comparative experiments on 20 common bacterial species in addition to the 13 NTMs. The specific detection steps were the same as in Example 4. All primers and probes tested negative for other NTMs, Mycobacterium tuberculosis, BCG, and common pneumonia-causing pathogens. This demonstrates that the primer and probe combination provided by the present invention for typing nontuberculous mycobacteria has good sensitivity and specificity for the target bacterial species.

[0057] Table 5

[0058] The abbreviations and Latin names of the 20 common bacterial species are: MPE: M. peregrinum (Mycobacterium exogenum); MTR: M. triplex (Triple Mycobacterium); MLE: M. lentiflavum (Mycobacterium lentiflavum); MBR: M. from Brisbane (Mycobacterium brisbane); MIR: M. iranicum (Mycobacterium spp. Irani); MMAG:M. from Magerita (Mycobacterium margaritiferum); MMUC: M. mucogenicum (Mycobacterium mucinigenes); MNA: M. new gold (Mycobacterium neogoldensis); MNC: M. novocastrense (Mycobacterium nematodeum); MOB: M. obverse (Mycobacterium aube); MSE: M. seoulense (Mycobacterium Seoulensis); MSHI: M. shimoidei (Mycobacterium); MTB: M. tuberculosis; BCG: M. ox BCG (Bacillus Calmette-Guérin); KP: Klebsiella pneumoniae (Klebsiella pneumoniae); S. aureus : Staphylococcus aureus (Staphylococcus aureus); PA: Pseudomonas aeruginosa (Pseudomonas aeruginosa); E. coli : Escherichia coli (Escherichia coli); C. neoformans : Cryptococcus neoformans (Cryptococcus neoformans); A. fumigatus : Aspergillus fumigatus (Aspergillus fumigatus).

[0059] The present invention is not limited to the specific embodiments disclosed and described above. Modifications and variations of the present invention without departing from the spirit and scope of the inventive concept should also fall within the scope of the claims of the present invention and be protected by the appended claims. Furthermore, although certain specific terms are used in this specification, these terms are for convenience only and do not constitute any limitation of the present invention.

Claims

1. A primer and probe combination for typing detection of non-tuberculous mycobacteria, characterized in that Including specific forward primers, reverse primers and probes for 13 non-tuberculous mycobacteria, including Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium massa, Mycobacterium abscessus, Mycobacterium chelonae, Mycobacterium kansasii, Mycobacterium fortuitum, Mycobacterium toadense, Mycobacterium marinum, Mycobacterium ulcerans, Mycobacterium gordonii, Mycobacterium paragordonii and Mycobacterium simianum; The specific forward primers, reverse primers and probe sequences of the 13 non-tuberculosis mycobacteria are shown in SEQ ID NOs: 1 to 39.

2. The primer and probe combination for typing detection of non-tuberculous mycobacteria according to claim 1, characterized in that: The sequences are the reverse complementary sequences of SEQ ID NOs: 1-39.

3. The primer and probe combination for typing detection of non-tuberculous mycobacteria according to claim 1, characterized in that: The 5' end of the probe sequence is labeled with a fluorescent group, and the 3' end is labeled with a quencher group.

4. The primer and probe composition for typing detection of 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; the quenching group is selected from any one of BHQ0, BHQ1, BHQ2, BHQ3 or MGB.

5. The primer and probe composition for typing detection of non-tuberculous mycobacteria according to claim 1, characterized in that: The concentration ratio of the 13 non-tuberculosis mycobacteria specific forward primers, reverse primers and probes is 1-5:1-5:1-4.

6. A method for detecting nontuberculous mycobacteria typing, characterized in that: The following steps are involved: S1: Use bacterial genomic DNA extraction reagent to extract bacterial genomic DNA from the sample to be tested and store it at -20~-80℃; S2: using the bacterial genomic DNA in the sample to be tested as a template, and performing PCR amplification using the primer and probe combination for non-tuberculosis mycobacterium typing detection according to any one of claims 1 to 5; S3: Interpret the PCR results based on the fluorescence signal.

7. The method for detecting nontuberculous mycobacteria typing according to claim 6, characterized in that: The PCR amplification reaction conditions in step S2 include: Stage 1: 35-39°C, 2-3 minutes; Stage 2: 94°C-96°C, 30s-60s; Stage 3: 94°C-96°C, 4s-10s; Stage 4: 55°C-60°C, 30s-60s; Stages 3 and 4 are repeated for 20-50 cycles.

8. The method for detecting nontuberculous mycobacteria typing according to claim 6, wherein: The judgment standard in step S3 is that a signal is detected at a Ct value of 35 or less.

9. A kit for the typing detection of non-tuberculous mycobacteria, characterized in that A primer and probe composition for detecting typing of non-tuberculous mycobacteria comprising the composition according to any one of claims 1 to 5.

10. Use of the primer and probe combination for typing detection of nontuberculous mycobacteria according to any one of claims 1 to 5, or the method for typing detection of nontuberculous mycobacteria according to any one of claims 6 to 8, or the typing detection kit according to claim 9 in the preparation of a product for typing detection of nontuberculous mycobacteria.

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