Primer probe combination and kit for identifying mycobacterium strains and identification method and application thereof

Abstract

The invention provides a primer probe combination and a kit for identifying a mycobacterium strain as well as an identification method and application of the primer probe combination and the kit, and belongs to the technical field of biology. The primer probe combination at least comprises one pair of primers and one probe for identifying mycobacterium avium and mycobacterium intracellularis, and six pairs of primers and six probes are respectively used for identifying mycobacterium abscessus, mycobacterium kansasii, mycobacterium accidental, mycobacterium marinum / mycobacterium ulcerasus, mycobacterium gordoni and mycobacterium scrofula. The two pairs of primers and the two probes are used for identifying the mycobacterium tuberculosis complex group, and the one pair of primers and the one probe are used for identifying the internal standard. According to the invention, a plurality of indexes (at most 9 indexes) can be synchronously detected by one sample, and the mycobacterium tuberculosis complex group and various non-mycobacterium tuberculosis can be identified at the same time; moreover, the detection sensitivity is high, the lowest detection limit of mycobacterium tuberculosis complex is 50 CFU / mL, and the lowest detection limit of nontuberculous mycobacteria is 500 CFU / mL.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a primer-probe combination, a kit and an identification method and application thereof for identifying mycobacterium species. Background Art

[0002] Tuberculosis and non-tuberculosis diseases caused by mycobacterium infection are public health problems worldwide. Tuberculosis is a chronic infectious disease caused by the Mycobacterium tuberculosis complex (MTBC). According to the "2023 Global Tuberculosis Report" released by the World Health Organization, in 2022, the estimated number of tuberculosis patients worldwide is 10.6 million, and about 1.3 million patients will die from tuberculosis. The number of new patients in my country is about 748,000, ranking third in the world.

[0003] Non-tuberculous mycobacteria (NTM) and MTBC belong to the genus Mycobacterium and are a large class of environmental bacteria that are widely found in soil, tap water or air. Some NTM can invade human organs and tissues and cause various diseases, mainly including lung disease, lymphadenitis, skin and soft tissue infections, and systemic disseminated diseases. People with immunodeficiency or immunodeficiency are more likely to be infected with non-tuberculous diseases and develop diseases. Among them, the clinical symptoms of NTM lung disease and pulmonary tuberculosis are similar, and diagnosis can only be made through species identification. However, traditional smear acid-fast staining and mycobacterial isolation and culture in tuberculosis laboratories cannot distinguish between Mycobacterium tuberculosis complex (MTBC) and NTM. If NTM lung disease is misdiagnosed as pulmonary tuberculosis and treated with anti-tuberculosis drugs, the treatment effect is often poor because NTM is inherently resistant to anti-tuberculosis drugs and there are differences in the treatment regimens for non-tuberculosis and pulmonary tuberculosis. In addition, different NTM strains have different sensitivities to drugs. Therefore, in order to guide the selection of clinical treatment options, the identification of mycobacterial species is of great significance.

[0004] In recent years, due to the long culture cycle, cumbersome operation and poor accuracy, traditional biochemical identification has been gradually replaced by molecular biological methods. At present, common molecular identification methods include linear probe analysis (LPA), real-time fluorescence PCR, matrix-assisted laser desorption tandem time-of-flight mass spectrometry (MALDI-TOF MS) and DNA Sanger sequencing. Among them, LPA requires post-PCR processing, there is a risk of contamination of the amplified product, the operation steps are cumbersome, and it takes at least 6 hours to produce results. Real-time fluorescence PCR is a method in which primer probes are designed for specific target genes of the sample to be tested, and detection is achieved through the change of fluorescence signals during the PCR reaction. Real-time fluorescence PCR has a short detection time, strong specificity and high sensitivity, but its detection throughput is often limited by the number of fluorescence channels. At present, most fluorescence PCR instruments on the market have only 4 to 6 channels, which limits the ability of real-time fluorescence PCR to detect multiple targets simultaneously in a single tube. In recent years, MALDI-TOF MS technology has developed rapidly and is widely used in the identification of bacterial infections, but it can only identify clinical isolates. Since most NTM are slow-growing bacteria, they require at least several weeks of isolation and culture before MALDI-TOF MS identification, which seriously affects the timeliness of this method. DNA Sanger sequencing is the "gold standard" for genetic testing methods, but it requires specialized sequencing equipment, which is expensive and cannot be widely used in clinical laboratories.

[0005] At present, there are relevant commercial kits for the identification of mycobacterium species, including the "Mycobacterium species identification gene detection kit (PCR-reverse dot hybridization method)" of Yaneng Biotechnology (Shenzhen) Co., Ltd. and the "Mycobacterium species identification kit (DNA microarray chip method)" of Chengdu Bio Jingxin Biotechnology Co., Ltd. The basic principle of these two kits is to hybridize the amplified product with the DNA probe and use the color development / luminescence reaction of the hybridization band to identify the mycobacterium strain. Compared with the sequencing method, this method simplifies the operation to a certain extent, so it has been further applied in clinical practice, but this method has certain limitations, such as poor detection sensitivity (10 5 bacteria / mL and 10 3 Based on the fluorescent PCR melting curve method, Xiamen Zhishan Biotechnology Co., Ltd. has developed the "Mycobacterium Identification Kit (Fluorescent PCR Melting Curve Method)", which has strong identification ability and can accurately identify 19 common clinical mycobacteria. However, this method is not a fully automated detection product, and the detection time is relatively long (4h). It cannot directly detect clinical sputum samples, but requires sputum samples to be cultured before detection, which still limits its application in clinical practice. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides a primer-probe combination, a kit and an identification method for identifying mycobacterium species, which can simultaneously identify multiple common clinical mycobacteria within 2 hours with good specificity and high detection sensitivity.

[0007] The primer-probe combination for identifying mycobacterial species of the present invention comprises one or more of the following primers and probes, respectively:

[0008] The nucleotide sequences of the primers for identifying Mycobacterium avium and Mycobacterium intracellulare are shown in SEQ ID NO.1 and SEQ ID NO.2, and the probe is shown in SEQ ID NO.3;

[0009] The nucleotide sequences of the primers for identifying Mycobacterium gordonii are shown in SEQ ID NO.4 and SEQ ID NO.5, and the probe is shown in SEQ ID NO.6;

[0010] The nucleotide sequences of the primers for identifying Mycobacterium scrofulae are shown in SEQ ID NO.7 and SEQ ID NO.8, and the probe is shown in SEQ ID NO.9;

[0011] The nucleotide sequences of the primers for identifying Mycobacterium fortuitum are shown in SEQ ID NO.10 and SEQ ID NO.11, and the probe is shown in SEQ ID NO.12;

[0012] The nucleotide sequences of the primers for identifying Mycobacterium abscessus are shown in SEQ ID NO. 13 and SEQ ID NO. 14, and the probe is shown in SEQ ID NO. 15;

[0013] The nucleotide sequences of the primers for identifying Mycobacterium kansasii are shown in SEQ ID NO. 16 and SEQ ID NO. 17, and the probe is shown in SEQ ID NO. 18;

[0014] The nucleotide sequences of the primers for identifying Mycobacterium marinum or Mycobacterium ulcerans are shown in SEQ ID NO.19 and SEQ ID NO.20, and the probe is shown in SEQ ID NO.21;

[0015] The method for identifying the Mycobacterium tuberculosis complex includes 2 pairs of primers and 2 probes. The nucleotide sequence of the first pair of primers is shown in SEQ ID NO.22 and SEQ ID NO.23, and the probe is shown in SEQ ID NO.24; the nucleotide sequence of the second pair of primers is shown in SEQ ID NO.25 and SEQ ID NO.26, and the probe is shown in SEQ ID NO.27.

[0016] Preferably, a pair of primers and probes for identifying internal standards are further included; the nucleotide sequences of the primers are shown in SEQ ID NO.28 and SEQ ID NO.29, and the probe is shown in SEQ ID NO.30.

[0017] More preferably, the detection probes for mycobacteria and internal standards are modified with a fluorescent group at the 5' end and a quenching group at the 3' end;

[0018] The fluorescent group is selected from any one of FAM, VIC, TET, JOE, HEX, CY3, CY5, TAMRA, ROX, Texas Red, CY5.5 or CY7;

[0019] The quenching group is selected from any one of BHQ0, BHQ1, BHQ2, BHQ3 or MGB.

[0020] The present invention also provides a kit for identifying mycobacterial species, wherein the kit comprises the primer-probe combination.

[0021] The present invention also provides a method for identifying mycobacterium species using the kit, comprising the following steps: (1) extracting nucleic acid from a sample to be tested; (2) performing PCR amplification using the extracted nucleic acid as a template, interpreting the result of the PCR amplification, and identifying the mycobacterium species in the sample to be tested.

[0022] Preferably, the PCR amplification system is: template 0.1-50 μL, PCR amplification reagent 5-50 μL; the PCR amplification reagent is prepared as: 2× amplification mix 1×; the primers are each 0.01-10 μM, and the probes are each 0.01-10 μM.

[0023] Preferably, the PCR amplification program is: pre-denaturation at 95°C for 1 to 3 min, denaturation at 95°C for 3 to 120 s, annealing and extension at 55 to 65°C for 5 to 100 s for a total of 40 to 60 cycles, denaturation at 95°C for 1 to 3 min, insulation at 35 to 45°C for 1 to 3 min, melting curve analysis at 40°C to 90°C, and a temperature increase of 0.1 to 10°C per step.

[0024] Preferably, the method of identifying the mycobacterium species is: identifying non-tuberculous mycobacteria by the Tm value of the specific melting curve of asymmetric PCR, and identifying the Mycobacterium tuberculosis complex by the Ct value of the amplification curve of symmetric PCR.

[0025] More preferably, when identifying non-tuberculous mycobacteria, the Tm value of Mycobacterium scrofulae is 65.0-69.0, the Tm value of Mycobacterium gordonii is 69.1-75.0, the Tm value of Mycobacterium fortuitum is 73.0-78.0, the Tm value of Mycobacterium abscessus is 65.0-76.0, and the Tm value of Mycobacterium avium is 63.0-78.0. 70.0, the Tm value judgment interval of intracellular mycobacterium is 70.1-76.0, the Tm value judgment interval of Mycobacterium kansasii is 66.0-73.0, the Tm value judgment interval of Mycobacterium marinum or Mycobacterium ulcerans is 66.0-78.0, and the Tm value judgment interval of the internal standard is 79.0-82.0; when identifying the Mycobacterium tuberculosis complex, the Ct of the Mycobacterium tuberculosis complex is ≤42.

[0026] The present invention also provides an application of the primer-probe combination or the kit in identifying mycobacterium species or preparing a reagent for identifying mycobacterium species.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] The primer-probe combination or kit of the present invention can realize the simultaneous detection of multiple indicators in one sample (up to 9 indicators can be detected simultaneously), and the Mycobacterium tuberculosis complex and 8 non-tuberculous mycobacteria can be identified simultaneously; and the detection sensitivity is high under the multiple system, the minimum detection limit of Mycobacterium tuberculosis complex is 50CFU / mL, and the minimum detection limit of non-tuberculous mycobacteria is 500CFU / mL. DETAILED DESCRIPTION

[0029] The present invention provides a primer-probe combination for identifying mycobacterium species, comprising at least one of the following 9 pairs of primers and 9 probes, wherein 1 pair of primers and 1 probe are used to identify mycobacterium avium and mycobacterium intracellulare, 6 pairs of primers and 6 probes are used to identify mycobacterium abscessus, mycobacterium kansasii, mycobacterium fortuitum, mycobacterium marinum / mycobacterium ulcerans, mycobacterium gordonii and mycobacterium scrofula, and 2 pairs of primers and 2 probes are used to identify mycobacterium tuberculosis complex; preferably, 1 pair of primers and 1 probe are further included for identifying an internal standard; more preferably, the 5' end of the detection probe of mycobacterium and the internal standard is modified with a fluorescent group, and the 3' end is modified with a quenching group; the fluorescent group is selected from FAM, VIC, TET, JOE, HEX, CY3, CY5, TAMRA, ROX, Texas Any one of Red, CY5.5 or CY7; the quenching group is selected from any one of BHQ0, BHQ1, BHQ2, BHQ3 or MGB.

[0030] In the present invention, the “ / ” used when expressing the results of strain identification represents the meaning of “or”. For example, “Mycobacterium marinum / Mycobacterium ulcerans” means that the strain can be identified as Mycobacterium marinum or Mycobacterium ulcerans, excluding other mycobacteria. Further identification can be carried out as needed to determine whether the strain is Mycobacterium marinum or Mycobacterium ulcerans.

[0031] Specifically, the specific sequences of primers and probes are shown in Table 1.

[0032] Table 1 Primer and probe sequences

[0033]

[0034]

[0035]

[0036] The present invention also provides a kit for identifying mycobacterial species, the kit comprising the primer-probe combination, i.e., asymmetric PCR restriction primers, non-restriction primers and corresponding probes, symmetric PCR upstream and downstream primers and probes, and preferably also comprising various other reagents required for nucleic acid sequence amplification, including but not limited to DNA polymerase, dNTPs, PCR buffer, etc. As an implementable embodiment, the above components are freeze-dried by vacuum freeze-drying technology, and after freeze-drying, they are present in the kit in the form of freeze-dried balls, and finally the freeze-dried balls are re-dissolved and amplified with the extracted nucleic acid template.

[0037] The present invention also provides a method for identifying mycobacterium species using the kit, which is based on a fluorescent quantitative PCR technology platform, combined with multiple asymmetric PCR technology and melting curve technology, and uses multiple fluorescence detection channels to detect melting peaks formed by multiple different fluorescently labeled probes and amplification products in one PCR reaction, and uses the Tm value shown by the melting peak to identify mycobacterium species, comprising the following steps: (1) extracting nucleic acid from a sample to be tested; (2) performing PCR amplification using the extracted nucleic acid as a template, interpreting the results of the PCR amplification, and identifying the mycobacterium species in the sample to be tested.

[0038] In the present invention, the preferred PCR amplification system is: template 0.1-50 μL, PCR amplification reagent 5-50 μL; the PCR amplification reagent is prepared as: 2× amplification mix 1×; the primers are 0.01-10 μM each, and the probes are 0.01-10 μM each; further preferably, the concentration of upstream and downstream primers of Mycobacterium tuberculosis complex group is 0.6 μM, the concentration of non-tuberculous mycobacterium limiting primer is 0.2 μM, the concentration of non-limiting primer is 4 μM; the probe concentration is 0.1 μM. More preferably, as shown in Table 2.

[0039] Table 2 PCR amplification system

[0040]

[0041] In the present invention, the preferred PCR amplification program is: 95°C pre-denaturation for 1-3 min, 95°C denaturation for 3-120 s, 55-65°C annealing extension for 5-100 s for a total of 40-60 cycles, 95°C denaturation for 1-3 min, 35-45°C insulation for 1-3 min, 40°C-90°C melting curve analysis, each step temperature increase of 0.1-10°C; more preferably 95°C pre-denaturation for 2 min, 95°C denaturation for 3-120 s, 55-65°C annealing extension for 5-120 s for a total of 40-60 cycles, 95°C denaturation for 2 min, 40°C insulation for 2 min, 40°C melting curve analysis for 40°C-90°C, each step temperature increase of 0.1-10°C. More preferably as shown in Table 3.

[0042] Table 3 PCR amplification program

[0043]

[0044] In the present invention, the preferred method for identifying mycobacterium species is to identify non-tuberculous mycobacteria by the Tm value of the specific melting curve of asymmetric PCR, and to identify the Mycobacterium tuberculosis complex by the Ct value of the amplification curve of symmetric PCR.

[0045] Analyzing the melting curve specifically means that due to the difference between the sequences of different bacterial species, the probe hybridizes with the sequences of different bacterial species to produce melting peaks with different Tm values, and the actually obtained Tm value is compared with the Tm value interval in Table 4, so as to identify the mycobacterial species. In the present invention, it is preferred that 8 non-tuberculous mycobacteria, including Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium abscessus, Mycobacterium kansasii, Mycobacterium fortuitum, Mycobacterium marinum / Mycobacterium ulcerans, Mycobacterium gordonii, and Mycobacterium scrofula, are identified by the specific melting curve Tm value generated after asymmetric PCR, while the Mycobacterium tuberculosis complex group is identified by symmetric PCR and observing the Ct value of the amplification curve. When further preferably identifying non-tuberculous mycobacteria, the Tm value of Mycobacterium scrofulae is 65.0-69.0, the Tm value of Mycobacterium gordonii is 69.1-75.0, the Tm value of Mycobacterium fortuitum is 73.0-78.0, the Tm value of Mycobacterium abscessus is 65.0-76.0, and the Tm value of Mycobacterium avium is 63.0-77.0. 0.0, the Tm value of intracellular mycobacterium is 70.1-76.0, the Tm value of kansasii is 66.0-73.0, the Tm value of marinum or ulcer mycobacterium is 66.0-78.0, and the Tm value of the internal standard is 79.0-82.0; when identifying the Mycobacterium tuberculosis complex, the Ct of the Mycobacterium tuberculosis complex is ≤42. The more preferred identification rules are shown in Table 4.

[0046] Table 4 Interpretation intervals of targets to be detected in each channel

[0047]

[0048]

[0049] The present invention also provides an application of the primer-probe combination or the kit in identifying mycobacterium species or preparing a reagent for identifying mycobacterium species.

[0050] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0051] Example 1

[0052] Screening of primer concentration

[0053] In this example, the concentration of primers in the amplification system was screened, including the concentration of upstream and downstream primers of symmetric PCR of Mycobacterium tuberculosis complex and the concentration of limiting primers and restriction primers of asymmetric PCR of non-tuberculosis Mycobacterium tuberculosis. One concentration was selected for each mycobacterium for verification (8 non-tuberculosis mycobacteria selected a concentration of 500 CFU / mL, and Mycobacterium tuberculosis selected a concentration of 50 CFU / mL). The detection values ​​under different primer concentrations are shown in Table 5: When the concentrations of upstream and downstream primers of Mycobacterium tuberculosis complex in the system were all 0.6 μM, the concentration of limiting primers of non-tuberculosis mycobacteria was 0.2 μM, and the concentration of non-restrictive primers was 4 μM, the Ct value of Mycobacterium tuberculosis was not significantly different from that of other conditions, and the positive rates of 8 non-tuberculosis mycobacteria were the highest (all 100%), so this condition was selected as the optimal concentration of primer probes in the system.

[0054] Table 5 Primer concentration screening for amplification system

[0055]

[0056]

[0057] Example 2

[0058] Simultaneous identification of nine mycobacterial species

[0059] The 8 non-tuberculous mycobacteria and Mycobacterium tuberculosis within the scope of the kit were mixed to prepare a mixed sample with a final concentration of 500 CFU / mL for non-tuberculous mycobacteria and 50 CFU / mL for Mycobacterium tuberculosis, and the test results were positive for 8 non-tuberculous mycobacteria and a positive Mycobacterium tuberculosis complex. As shown in Table 6, the mycobacterium identification reagent of the present invention can quickly and accurately identify 9 mycobacteria at the same time, and can identify mixed infections of two or more mycobacteria.

[0060] Table 6 Detection results when 9 species of mycobacteria coexist

[0061] Test content Ct value Tm value Test results Mycobacterium gordonii / 73.7 Positive Mycobacterium scrofula / 67.9 Positive Mycobacterium fortuitum / 75.1 Positive Mycobacterium abscessus / 73.2 Positive Mycobacterium avium / 68.2 Positive Mycobacterium intracellulare / 74.1 Positive Mycobacterium tuberculosis complex 38.91 / Positive Internal standard (Bacillus subtilis) 32.55 79.8 Positive Mycobacterium kansasii / 68.8 Positive Mycobacterium marinum / Mycobacterium ulcerans / 68.6 /

[0062] Example 3

[0063] Minimum detection limit verification

[0064] The process is as follows: prepare the minimum detection limit concentration under the negative sputum matrix, prepare 8 non-tuberculous mycobacteria at 500 CFU / mL and Mycobacterium tuberculosis at 50 CFU / mL, and make 10 parallels for each, which are used for full-process detection. At the same time, negative sputum without mycobacterium template is used as a negative control.

[0065] The minimum detection limit verification experiment is shown in Table 7: when the concentration of non-tuberculous mycobacteria is 500 CFU / mL, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium abscessus, Mycobacterium kansasii, Mycobacterium fortuitum, Mycobacterium sea / ulcerans, Mycobacterium gordonii, and Mycobacterium scrofula all have corresponding melting peaks in the corresponding channels, and are 100% positive; and when the concentration of Mycobacterium tuberculosis is 50 CFU / mL, Mycobacterium tuberculosis complex group has amplification peaks in the corresponding channels, and is 100% positive, and the remaining non-tuberculous mycobacteria have no corresponding melting peaks. Therefore, the minimum detection limit of the mycobacterium identification reagent of the present invention is 50 CFU / mL for Mycobacterium tuberculosis complex group and 500 CFU / mL for non-tuberculous mycobacteria.

[0066] Table 7 Verification results of minimum detection limit of 9 mycobacteria

[0067]

[0068]

[0069]

[0070] Example 4

[0071] Repeatability test of the kit

[0072] The weak positive samples of Mycobacterium tuberculosis (75 CFU / mL) and 8 weak positive samples of non-tuberculous mycobacteria (750 CFU / mL) diluted with negative sputum matrix were repeated 10 times, and the CV of the Ct value of Mycobacterium tuberculosis and the CV of the Tm value of 8 non-tuberculous mycobacteria were calculated. As shown in Table 8, the CV of the Ct value was ≤5%, and the CV of the Tm value was ≤2%.

[0073] Table 8 Intra-assay and inter-assay precision CV of Ct / Tm values ​​of different samples

[0074]

[0075]

[0076] Example 5

[0077] Kit specificity test

[0078] According to the cross-reaction evaluation method, 20 bacterial or fungal cross-reactants, including Mycobacterium terrestris, Mycobacterium minor, Mycobacterium toad, Mycobacterium suga, Mycobacterium chelonae, Mycobacterium smegmatis, Mycobacterium gastricis, Mycobacterium phlei, Mycobacterium malmoe, Mycobacterium achromogenes, Mycobacterium simianus, Streptococcus pneumoniae, Haemophilus influenzae, Escherichia coli, Staphylococcus epidermidis, Cryptococcus, Staphylococcus aureus, Nocardia spp., Pseudomonas aeruginosa, and Candida albicans, were diluted to 10% with negative sputum matrix. 6 CFU / mL, the five cross-reacting viruses, novel influenza A (H1N1) (2009) virus (human influenza virus type A), influenza B virus Victoria strain (human influenza virus type B), human parainfluenza virus (type 1), human parainfluenza virus (type 2), and human parainfluenza virus (type 3), were diluted to 10 in negative sputum matrix. 5 PFU / mL, 25 pathogens were detected, and the kit of the present invention was used for extraction and the fluorescence PCR+melting curve method was used for detection, and the detection results were all negative, indicating that the mycobacterium identification reagent of the present invention has no cross-reaction with the above pathogens and has good specificity.

[0079] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A primer-probe combination for identifying mycobacterial species, characterized in that: It includes one or more of the following primers and probes: The nucleotide sequences of the primers for identifying Mycobacterium avium and Mycobacterium intracellulare are shown in SEQ ID NO.1 and SEQ ID NO.2, and the probe is shown in SEQ ID NO.3; The nucleotide sequences of the primers for identifying Mycobacterium gordonii are shown in SEQ ID NO.4 and SEQ ID NO.5, and the probe is shown in SEQ ID NO.6; The nucleotide sequences of the primers for identifying Mycobacterium scrofulae are shown in SEQ ID NO.7 and SEQ ID NO.8, and the probe is shown in SEQ ID NO.9; The nucleotide sequences of the primers for identifying Mycobacterium fortuitum are shown in SEQ ID NO.10 and SEQ ID NO.11, and the probe is shown in SEQ ID NO.12; The nucleotide sequences of the primers for identifying Mycobacterium abscessus are shown in SEQ ID NO. 13 and SEQ ID NO. 14, and the probe is shown in SEQ ID NO. 15; The nucleotide sequences of the primers for identifying Mycobacterium kansasii are shown in SEQ ID NO. 16 and SEQ ID NO. 17, and the probe is shown in SEQ ID NO. 18; The nucleotide sequences of the primers for identifying Mycobacterium marinum or Mycobacterium ulcerans are shown in SEQ ID NO.19 and SEQ ID NO.20, and the probe is shown in SEQ ID NO.21; The method for identifying the Mycobacterium tuberculosis complex includes 2 pairs of primers and 2 probes. The nucleotide sequence of the first pair of primers is shown in SEQ ID NO.22 and SEQ ID NO.23, and the probe is shown in SEQ ID NO.24; the nucleotide sequence of the second pair of primers is shown in SEQ ID NO.25 and SEQ ID NO.26, and the probe is shown in SEQ ID NO.

27.

2. The primer-probe combination according to claim 1, characterized in that: It also includes a pair of primers and a probe for identifying an internal standard; the nucleotide sequences of the primers are shown in SEQ ID NO.28 and SEQ ID NO.29, and the probe is shown in SEQ ID NO.

30.

3. The primer-probe combination according to claim 1 or 2, characterized in that: The detection probes for mycobacteria and internal standards are modified with a fluorescent group at the 5' end and a quenching group at the 3' end; 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.

4. A kit for identifying mycobacterium species, characterized in that: The kit comprises the primer-probe combination according to any one of claims 1 to 3.

5. A method for identifying mycobacterial species using the kit according to claim 4, characterized in that: The following steps are involved: (1) extracting nucleic acid from the sample to be tested; (2) performing PCR amplification using the extracted nucleic acid as a template, interpreting the results of the PCR amplification, and identifying the mycobacterium species in the sample to be tested.

6. The method according to claim 5, characterized in that The PCR amplification system is: template 0.1-50 μL, PCR amplification reagent 5-50 μL; the PCR amplification reagent is prepared as: 2× amplification mix 1×; the primers described in claim 1 and claim 2 are each 0.01-10 μM, and the probes are each 0.01-10 μM.

7. The method according to claim 5, characterized in that The PCR amplification procedure is: pre-denaturation at 95°C for 1 to 3 minutes, denaturation at 95°C for 3 to 120 seconds, annealing and extension at 55 to 65°C for 5 to 100 seconds for a total of 40 to 60 cycles, denaturation at 95°C for 1 to 3 minutes, insulation at 35 to 45°C for 1 to 3 minutes, melting curve analysis at 40°C to 90°C, and a temperature increase of 0.1 to 10°C per step.

8. The method according to claim 5, characterized in that The method for identifying mycobacterium species is: identifying non-tuberculosis mycobacteria through the Tm value of the specific melting curve of asymmetric PCR, and identifying the Mycobacterium tuberculosis complex group through the Ct value of the amplification curve of symmetric PCR.

9. The method according to claim 8, characterized in that When identifying non-tuberculous mycobacteria, the Tm value of Mycobacterium scrofulae is judged in the interval of 65.0-69.0, the Tm value of Mycobacterium Gordonii is judged in the interval of 69.1-75.0, the Tm value of Mycobacterium fortuitum is judged in the interval of 73.0-78.0, the Tm value of Mycobacterium abscessus is judged in the interval of 65.0-76.0, the Tm value of Mycobacterium avium is judged in the interval of 63.0-70.0, the Tm value of Mycobacterium intracellulare is judged in the interval of 70.1-76.0, the Tm value of Mycobacterium kansasii is judged in the interval of 66.0-73.0, the Tm value of Mycobacterium marinum or Mycobacterium ulcerans is judged in the interval of 66.0-78.0, and the Tm value of the internal standard is judged in the interval of 79.0-82.0; when identifying the Mycobacterium tuberculosis complex, the Ct of the Mycobacterium tuberculosis complex is ≤42.

10. Use of the primer-probe combination according to any one of claims 1 to 3 or the kit according to claim 4 in identifying mycobacterium species or preparing a reagent for identifying mycobacterium species.

Images