A primer-probe combination for identifying mycobacterial species, a kit, and an identification method and application thereof

Abstract

The present invention provides a primer-probe combination, a kit, an identification method and an application thereof for identifying mycobacterium species, and belongs to the field of biotechnology. The primer-probe combination of the present invention includes at least one pair of primers and one probe for identifying Mycobacterium avium and Mycobacterium intracellulare, six pairs of primers and six probes for identifying Mycobacterium abscessus, Mycobacterium kansasii, Mycobacterium fortuitum, Mycobacterium marinum / Mycobacterium ulcerans, Mycobacterium gordonii and Mycobacterium scrofulae, two pairs of primers and two probes for identifying Mycobacterium tuberculosis complex, and one pair of primers and one probe for identifying an internal standard. The present invention can realize the simultaneous detection of multiple indicators (up to 9 indicators) in one sample, and simultaneously identify Mycobacterium tuberculosis complex and a variety of non-tuberculosis mycobacteria; and the detection sensitivity is high, the minimum detection limit of Mycobacterium tuberculosis complex is 50 CFU / mL, and the minimum detection limit of non-tuberculosis mycobacteria is 500 CFU / mL.

Description

Technical Field

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

[0002] Tuberculosis and non-tuberculosis diseases caused by mycobacterial infection are global public health issues. Tuberculosis is a chronic infectious disease caused by the Mycobacterium tuberculosis complex (MTBC). According to the World Health Organization's 2023 Global Tuberculosis Report, in 2022, the estimated global incidence of tuberculosis was 10.6 million, with approximately 1.3 million deaths. my country had approximately 748,000 new cases, ranking third globally.

[0003] Non-tuberculous mycobacteria (NTM), belonging to the same genus as MTBC, are a large group of environmental bacteria found widely in soil, tap water, and air. Some NTM can invade human organs and tissues, causing a variety of diseases, including lung disease, lymphadenitis, skin and soft tissue infections, and systemic disseminated disease. Immunodeficiency or immunocompromise makes individuals more susceptible to non-tuberculous infections and disease. 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 are inherently resistant to anti-tuberculosis drugs, and treatment regimens for non-tuberculosis and pulmonary tuberculosis differ. Furthermore, different NTM strains exhibit varying drug susceptibility. Therefore, in order to guide the selection of clinical treatment options, the identification of mycobacterial species is of great significance.

[0004] In recent years, traditional biochemical identification has been gradually replaced by molecular biology methods due to their long culture cycles, cumbersome procedures, and poor accuracy. Currently, common molecular identification methods include line probe analysis (LPA), real-time fluorescence PCR, matrix-assisted laser desorption tandem time-of-flight mass spectrometry (MALDI-TOF MS), and DNA Sanger sequencing. LPA requires post-PCR processing, which carries the risk of contamination of the amplified product. The procedure is cumbersome and requires at least six hours to produce results. Real-time fluorescence PCR involves designing primers and probes specific to the target gene in the sample being tested, and detecting the gene by changes in the fluorescence signal during the PCR reaction. Real-time fluorescence PCR offers fast detection times, strong specificity, and high sensitivity, but its detection throughput is often limited by the number of fluorescence channels. Most commercially available fluorescence PCR instruments have only four to six channels, which limits the ability to simultaneously detect multiple targets in a single tube. In recent years, MALDI-TOF MS technology has developed rapidly and is widely used for the identification of bacterial infections, but it can only identify clinical isolates. Because most NTM are slow-growing bacteria, they require at least several weeks of isolation and culture before MALDI-TOF MS identification, which severely impacts the timeliness of this method. DNA Sanger sequencing is the gold standard for genetic testing, but it requires specialized sequencing equipment, which is expensive and not widely available 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, and therefore 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 capabilities and can accurately identify 19 common clinical mycobacteria. However, this method is not a fully automated detection product, the detection time is relatively long (4 hours), and it cannot be directly tested on clinical sputum samples. Instead, the sputum samples must be cultured before testing, 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 Mycobacterium tuberculosis complex includes two pairs of primers and two 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 an internal standard 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 quencher 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 mycobacterium species, which 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 results 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 0.01-10 μM each, and the probes are 0.01-10 μM each.

[0023] Preferably, the PCR amplification program 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 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-tuberculosis 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- The Tm value interpretation range of Mycobacterium intracellulare is 70.1-76.0, the Tm value interpretation range of Mycobacterium kansasii is 66.0-73.0, the Tm value interpretation range of Mycobacterium marinum or Mycobacterium ulcerans is 66.0-78.0, and the Tm value interpretation range 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 a use 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 simultaneously identify the Mycobacterium tuberculosis complex and 8 types of non-tuberculosis mycobacteria; and the detection sensitivity is high under the multiple system, with the minimum detection limit of Mycobacterium tuberculosis complex being 50 CFU / mL and the minimum detection limit of non-tuberculosis mycobacteria being 500 CFU / 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 scrofulae, 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 detection probes for mycobacteria and the internal standard are modified with a fluorescent group at the 5' end and a quenching group at the 3' end; 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 bacterial species 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 exactly 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 Mycobacterium species, comprising the aforementioned primer-probe combination, namely, asymmetric PCR restriction primers, non-restriction primers and corresponding probes, and symmetric PCR upstream and downstream primers and probes. Furthermore, the kit preferably includes various other reagents required for nucleic acid sequence amplification, including but not limited to DNA polymerase, dNTPs, and PCR buffer. In one embodiment, the aforementioned components are freeze-dried using vacuum freeze-drying technology and present in the kit as freeze-dried pellets. The pellets are then reconstituted and amplified using an extracted nucleic acid template.

[0037] The present invention also provides a method for identifying mycobacterial species using the kit, which is based on a fluorescent quantitative PCR technology platform, combines multiple asymmetric PCR technology and melting curve technology, and uses multiple fluorescence detection channels to detect melting peaks formed by multiple fluorescently labeled probes and amplified products in one PCR reaction, and uses the Tm value shown by the melting peak to identify mycobacterial 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 mycobacterial species in the sample to be tested.

[0038] In the present invention, the preferred PCR amplification system is: 0.1-50 μL template, 5-50 μL PCR amplification reagent; the PCR amplification reagent is prepared as follows: 2× amplification mix 1×; the primers are each 0.01-10 μM, and the probes are each 0.01-10 μM. Furthermore, the concentration of the upstream and downstream primers for the Mycobacterium tuberculosis complex is 0.6 μM, the concentration of the non-tuberculosis mycobacterium restrictive primer is 0.2 μM, and the concentration of the non-restrictive primer is 4 μM; and the probe concentration is 0.1 μM. More preferred is 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 and extension for 5-100 s for a total of 40-60 cycles, 95°C denaturation for 1-3 min, 35-45°C incubation for 1-3 min, 40-90°C melting curve analysis, with a temperature increase of 0.1-10°C per step; more preferably, 95°C pre-denaturation for 2 min, 95°C denaturation for 3-120 s, 55-65°C annealing and extension for 5-120 s for a total of 40-60 cycles, 95°C denaturation for 2 min, 40°C incubation for 2 min, 40°C melting curve analysis, with a temperature increase of 0.1-10°C per step. More preferably, the program is shown in Table 3.

[0042] Table 3 PCR amplification program

[0043]

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

[0045] Melting curve analysis specifically refers to the following: due to differences in sequences between different bacterial species, hybridization of the probe with the sequences of different bacterial species produces melting peaks with different Tm values. The actual Tm values ​​obtained are compared with the Tm value ranges in Table 4 to identify the mycobacterial species. In the present invention, eight non-tuberculosis mycobacteria, including Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium abscessus, Mycobacterium kansasii, Mycobacterium fortuitum, Mycobacterium marinum / Mycobacterium ulcerans, Mycobacterium gordonii, and Mycobacterium scrofulae, are preferably identified by the specific melting curve Tm values ​​generated after asymmetric PCR, while the Mycobacterium tuberculosis complex is identified by observing the Ct values ​​of the amplification curves using symmetric PCR. Furthermore, when identifying non-tuberculous mycobacteria, the Tm value of Mycobacterium scrofulae is preferably within the range of 65.0 to 69.0, the Tm value of Mycobacterium gordonii is within the range of 69.1 to 75.0, the Tm value of Mycobacterium fortuitum is within the range of 73.0 to 78.0, the Tm value of Mycobacterium abscessus is within the range of 65.0 to 76.0, and the Tm value of Mycobacterium avium is within the range of 63.0 to 77.0. The Tm value of the internal standard is 79.0-82.0. When identifying Mycobacterium tuberculosis complex, the Ct value of Mycobacterium tuberculosis complex is ≤ 42. 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 a use 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 with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0051] Example 1

[0052] Screening of primer concentration

[0053] In this example, the concentrations of primers in the amplification system were screened, including the concentrations of upstream and downstream primers for symmetric PCR of the Mycobacterium tuberculosis complex and the concentrations of restriction primers and limiting primers for asymmetric PCR of non-tuberculosis mycobacteria. One concentration was selected for each mycobacterium for validation (500 CFU / mL for eight non-tuberculosis mycobacteria and 50 CFU / mL for Mycobacterium tuberculosis). The detection values ​​at different primer concentrations are shown in Table 5. When the concentrations of upstream and downstream primers for the Mycobacterium tuberculosis complex were both 0.6 μM, the concentration of the restriction primer for non-tuberculosis mycobacteria was 0.2 μM, and the concentration of the non-restrictive primer was 4 μM, the Ct value for Mycobacterium tuberculosis was no significantly different from the other conditions, and the positive rate for all eight non-tuberculosis mycobacteria was the highest (all 100%). Therefore, this condition was selected as the optimal primer and probe concentration for this system.

[0054] Table 5 Primer concentration screening for amplification system

[0055]

[0056]

[0057] Example 2

[0058] Simultaneous identification of nine mycobacterial species

[0059] Eight nontuberculous mycobacteria in the kit were mixed with Mycobacterium tuberculosis to create a mixed sample with a final concentration of 500 CFU / mL for nontuberculous mycobacteria and 50 CFU / mL for Mycobacterium tuberculosis. The sample was then tested, and the results were positive for all eight nontuberculous mycobacteria and Mycobacterium tuberculosis complex. As shown in Table 6, the mycobacterium identification reagent of the present invention can rapidly and accurately identify nine mycobacteria simultaneously, and can also identify mixed infections with 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 scrofulae / 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 in the negative sputum matrix, prepare 8 non-tuberculosis 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] Table 7 shows the minimum detection limit validation experiment: at a non-tuberculous mycobacterium concentration of 500 CFU / mL, Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium abscessus, Mycobacterium kansasii, Mycobacterium fortuitum, Mycobacterium marinum / ulcerans, Mycobacterium gordonii, and Mycobacterium scrofula all exhibited corresponding melting peaks in the corresponding channels, with 100% positive results. Meanwhile, at a Mycobacterium tuberculosis concentration of 50 CFU / mL, Mycobacterium tuberculosis complex exhibited amplification peaks in the corresponding channels, with 100% positive results, while no corresponding melting peaks were observed for the remaining non-tuberculous mycobacteria. Therefore, the minimum detection limit of the mycobacterium identification reagent of the present invention is 50 CFU / mL for Mycobacterium tuberculosis complex and 500 CFU / mL for non-tuberculous mycobacteria.

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

[0067]

[0068]

[0069]

[0070] Example 4

[0071] Repeatability test of the kit

[0072] A weakly positive sample of Mycobacterium tuberculosis (75 CFU / mL) and eight weakly positive samples of nontuberculous mycobacteria (750 CFU / mL) diluted in negative sputum matrix were replicated 10 times. The CVs of the Ct values ​​for Mycobacterium tuberculosis and the Tm values ​​for the eight nontuberculous mycobacteria were calculated. As shown in Table 8, the CVs for the Ct values ​​were all ≤5%, and the CVs for the Tm values ​​were all ≤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 simiana, 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 virus cross-reactants 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 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. 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 principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. A primer-probe combination for identifying mycobacterial species, characterized in that: Includes 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 Mycobacterium tuberculosis complex includes two pairs of primers and two probes. The nucleotide sequences of the first pair of primers are shown in SEQ ID NO. 22 and SEQ ID NO. 23, and the probe is shown in SEQ ID NO.

24. The nucleotide sequences of the second pair of primers are shown in SEQ ID NO. 25 and SEQ ID NO. 26, and the probe is shown in SEQ ID NO.

27. 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.

2. The primer-probe combination according to claim 1, 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.

3. A kit for identifying mycobacterium species, characterized in that: The kit comprises the primer-probe combination according to claim 1 or 2.

4. A PCR amplification system for identifying mycobacterial species, characterized in that: The PCR amplification system is: 0.1-50 μL of template, 5-50 μL of PCR amplification reagent, 0.01-10 μM of each primer of claim 1, and 0.01-10 μM of each probe of claim 1; the PCR amplification reagent is: 2× amplification mix, and the reaction system concentration is 1×.

5. Use of the primer-probe combination according to claim 1 or 2 or the kit according to claim 3 in preparing a reagent for identifying mycobacterium species.

Images