Method for detecting mycobacterium tuberculosis and non-tuberculosis and application thereof

Abstract

This invention discloses a method for detecting tuberculous and non-tuberculous mycobacteria and its application, belonging to the field of biodetection technology. Addressing the combined problems of poor specificity, limited detection targets, high equipment costs, and long detection cycles in existing tuberculous and non-tuberculous mycobacteria detection technologies, this invention achieves specific detection by utilizing the ligation specificity of ligases. Ligases are highly precise; if a single base cannot be correctly matched, the ligation reaction will fail.

Description

Technical Field

[0001] This invention belongs to the field of biological detection technology, specifically relating to a method for detecting tuberculous and non-tuberculous mycobacteria and its application. Background Technology

[0002] Tuberculosis is caused by the Mycobacterium tuberculosis complex. Chronic infectious diseases caused by infection can affect the lungs and multiple other organs. Non-tuberculous mycobacteria (NTM) refers to all mycobacteria except for the Mycobacterium tuberculosis complex and Mycobacterium leprae. Patients with NTM lung disease and those with pulmonary tuberculosis both have lung lesions and similar clinical presentations, making differentiation difficult and often leading to misdiagnosis as pulmonary tuberculosis. However, the treatment regimens and courses for NTM and Mycobacterium tuberculosis are significantly different.

[0003] Currently, traditional methods for detecting tuberculous and non-tuberculous mycobacteria mainly rely on bacterial culture. However, mycobacteria grow slowly, requiring a long culture period, generally 2-6 weeks or even longer, which is insufficient to meet the needs of rapid clinical diagnosis. Furthermore, culture methods demand sophisticated laboratory conditions, requiring specialized technicians and equipment, and pose certain biosafety risks. Microscopic examination involves observing the morphological characteristics of mycobacteria through acid-fast staining, but this method has low sensitivity, is prone to false negatives, and cannot distinguish between tuberculous and non-tuberculous mycobacteria. Immunological detection methods, such as enzyme-linked immunosorbent assays (ELISA), detect specific antibodies or antigens against mycobacteria in the patient's body; however, these methods suffer from poor specificity, susceptibility to cross-reaction interference, and may yield inaccurate results in patients with weakened or suppressed immune systems. With the development of molecular biology, various nucleic acid detection technologies have emerged in the market. For example, fluorescent PCR technology is widely used for the detection of tuberculous and non-tuberculous mycobacteria. Fluorescent PCR technology has high sensitivity and can detect the nucleic acid sequence of mycobacteria, but it has certain limitations, such as only being able to detect specific mycobacterial species, having a limited range of detectable species, being unable to detect both tuberculous and non-tuberculous mycobacteria simultaneously, and having certain specificity issues. Next-generation sequencing technology has high detection costs, long detection cycles, and expensive sequencing equipment, which also limits its widespread application in the detection of tuberculous and non-tuberculous mycobacteria.

[0004] Given the limitations of the above technologies, there is an urgent need to develop a new detection method that can simultaneously detect tuberculous and non-tuberculous mycobacteria. Summary of the Invention

[0005] To address the combined problems of poor specificity, limited detection targets, high equipment costs, and long detection cycles in existing detection technologies for tuberculous and non-tuberculous mycobacteria, this invention provides a method for detecting tuberculous and non-tuberculous mycobacteria and its application, as well as a reagent for detecting tuberculous and non-tuberculous mycobacteria and its application in nucleic acid detection. Specificity of detection is achieved by utilizing the ligation specificity of ligases. Ligases are highly precise; if a single base cannot be correctly matched, the ligation reaction will fail.

[0006] The technical principle of this invention is as follows:

[0007] (1) The upstream probe and the downstream probe hybridize and bind to the single-stranded template, respectively;

[0008] (2) When the upstream and downstream probes hybridize and bind to the single-stranded template respectively, they are accurately complementary and there is no gap. The ligase catalyzes the formation of a phosphodiester bond between the 5' phosphate end of the downstream probe and the 3' hydroxyl end of the upstream probe.

[0009] (3) The upstream primer and the downstream primer bind complementaryly to the upstream primer complementary region and the downstream primer complementary region of the upstream probe and the downstream probe, respectively. Since the concentration of the downstream primer is higher than that of the upstream primer, asymmetric amplification is formed, generating a large amount of single-stranded DNA with a biotinylate group modified at the 5' end.

[0010] (4) Single-stranded DNA with a biotinylated group at the 5' end hybridizes with the corresponding target probes fixed on the vector. The target sites are detected by spot colorimetric analysis using the biotin-streptavidin-enzyme reaction system.

[0011] (5) When the upstream and downstream probes cannot hybridize and bind to the single-stranded template respectively, or cannot accurately pair up after binding, and there is a gap, the ligase cannot catalyze the formation of a phosphodiester bond between the 5' phosphate end of the downstream probe and the 3' hydroxyl end of the upstream probe;

[0012] (6) The upstream primer and the downstream primer cannot bind complementaryly to the upstream primer complementary region and the downstream primer complementary region of the upstream probe and the downstream probe, respectively, so amplification or asymmetric amplification cannot be achieved, and thus a single-stranded DNA with a biotinylate group modified at the 5' end cannot be generated.

[0013] (7) Because it is impossible to generate single-stranded DNA with a biotinylate group modified at the 5' end, when hybridizing with the corresponding target probe fixed on the vector, the biotin-streptavidin-enzyme reaction system does not produce spot color development.

[0014] The objective of this invention is achieved through the following technical solution:

[0015] A method for detecting tuberculous and nontuberculous mycobacteria and its application: A ligase can catalyze the formation of a phosphodiester bond between the 5' phosphate end and the 3' hydroxyl end of two adjacent DNA or RNA strands. These two DNA or RNA strands need to hybridize and accurately pair with complementary DNA or RNA strands without gaps, so as to achieve the detection of each detection target.

[0016] The ligases mentioned include, but are not limited to, commercially available Taq DNA Ligase, Ligase-65, 9°N DNA Ligase, HiFi Taq DNA Ligase, etc.

[0017] The method for detecting tuberculous and non-tuberculous mycobacteria and its application comprises an upstream probe, a downstream probe, an upstream primer, a downstream primer, a complementary region of the upstream primer, an upstream linker region, a downstream linker region, a complementary region of the downstream primer, and a target probe, thereby enabling the detection of each target point.

[0018] The upstream probe is a synthetically produced single-stranded DNA, single-stranded RNA, or single-stranded RNA-DNA with a length between 30 nt and 80 nt. It consists of two parts: an upstream primer complementary region and an upstream linker region. The upstream linker region has a length between 20 nt and 45 nt and is complementary to the template sequence.

[0019] The downstream probe is a synthetically produced single-stranded DNA, single-stranded RNA, or single-stranded DNA-RNA sequence, with a length between 30 and 80 nt. The 5' end of the sequence is modified with a phosphate group, and it consists of a downstream linker region and a downstream primer complementary region. The downstream linker region is 20 to 45 nt in length and is complementary to the template sequence.

[0020] The upstream primer is a synthetic single-stranded DNA, single-stranded RNA, or single-stranded DNA-RNA with a length between 15 nt and 55 nt, and has the same or partially the same sequence as the complementary region of the upstream primer.

[0021] The downstream primer is a synthetic single-stranded DNA, single-stranded RNA, or single-stranded DNA-RNA with a length between 15 nt and 55 nt. The 5' end of the sequence is modified with biotin or digoxigenin groups, and the sequence is completely or partially complementary to the complementary region of the downstream primer.

[0022] Each detection target point has a unique target probe that is either identical to or complementary to the connection sequence of the upstream connection region + downstream connection region.

[0023] For each detection target, upstream and downstream primers with the same or different sequences can be selected.

[0024] Specifically, a method for detecting tuberculous and non-tuberculous mycobacteria includes the following steps:

[0025] S1. Membrane chip fabrication:

[0026] The target probe and hybridization probe were diluted to 50 μM and fixed according to the probe immobilization kit instructions to obtain the membrane chip.

[0027] S2. Preparation of probe mixture:

[0028] Dilute all upstream and downstream probes to a concentration of 1 μM, take 1.2 μL of each and mix them together, then add 600 μL of 1×TE solution to obtain probe mixture; take 1.5 µL of probe mixture, 5 µL of 1×TE solution, and 1.5 µL of the above sample DNA and mix them together in a PCR tube.

[0029] S3, denaturation, hybridization, and ligation reactions:

[0030] Set the PCR instrument program, run the above PCR tubes through the denaturation and hybridization reaction program, and obtain the hybridization product;

[0031] Add 2 µL of hybridization product to the corresponding PCR tube, cap the tube, place the PCR tube in the PCR instrument, run the ligation reaction program, and obtain the ligation product.

[0032] S4, PCR amplification:

[0033] Dilute the upstream and downstream primers to a concentration of 10 μM, and prepare PCR reaction systems according to the formula to meet the required number of experiments; S

[0034] S5, Membrane Chip Detection:

[0035] Furthermore, the denaturation and hybridization reaction procedure described in S3 is as follows:

[0036]

[0037] Furthermore, in S3, a connecting mixture needs to be prepared:

[0038]

[0039] Furthermore, the connection reaction procedure described in S3 has the following parameters:

[0040]

[0041] Furthermore, the PCR amplification described in S4 involves the following preparation of the PCR reaction system:

[0042]

[0043] Furthermore, the PCR amplification described in S4, wherein the PCR instrument program is set as follows:

[0044]

[0045] This invention utilizes ligase to specifically ligate upstream and downstream probes complementary to the template, integrating them into a single unit. After asymmetric amplification and enrichment of the biotin-modified target sequence, it hybridizes with the target probe immobilized on a vector. Enzymatic display enables a detection method combining multiplexed probe amplification and membrane chip assays, achieving rapid and specific detection of both tuberculous and non-tuberculous mycobacteria. This method offers high specificity, high sensitivity, low cost, and eliminates the need for expensive equipment. Compared to existing technologies, this invention offers the following advantages:

[0046] (1) Reduced testing costs: Current mycobacterial identification technologies, such as gene chips and mNGS, have a single test reagent cost of hundreds to thousands of yuan, and require the purchase of testing equipment worth millions of yuan, making it difficult to promote at the grassroots level. The present invention reduces the cost of a single test reagent by more than 80%, requiring only a common nucleic acid amplification instrument.

[0047] (2) Improved detection speed: Traditional culture and identification takes 4-6 weeks and cannot identify non-tuberculous mycobacterial species; gene chip detection takes 6 hours and next-generation sequencing takes about 48 hours, while this invention can be completed within 2 hours. Attached Figure Description

[0048] Figure 1 This is a technical schematic diagram of a method for detecting tuberculous and non-tuberculous mycobacteria according to the present invention;

[0049] Figure 2 This is a graph showing the results of DNA testing for Mycobacterium tuberculosis in a clinical sample.

[0050] Figure 3 This is a graph showing the detection results of cultured Mycobacterium tuberculosis DNA diluted to a concentration of 0.1 pg / μL;

[0051] Figure 4 This is a diagram showing the results of DNA testing on a clinical sample of Mycobacterium avium.

[0052] Figure 5 This is a graph showing the detection results of cultured Mycobacterium avium DNA diluted to a concentration of 0.1 pg / μL;

[0053] Figure 6 This is a graph showing the DNA test results of a clinical sample of Mycobacterium Kansas.

[0054] Figure 7This is a graph showing the detection results of cultured Mycobacterium Kansas DNA diluted to a concentration of 0.1 pg / μL;

[0055] Figure 8 This is a graph showing the DNA test results of a clinical sample of Mycobacterium abscessus;

[0056] Figure 9 This is a graph showing the detection results of cultured Mycobacterium abscessus DNA diluted to a concentration of 0.1 pg / μL;

[0057] Figure 10 This is a graph showing the detection results of intracellular mycobacterial DNA in a clinical sample;

[0058] Figure 11 This is a graph showing the detection results of cultured intracellular mycobacterial DNA diluted to a concentration of 0.1 pg / μL;

[0059] Figure 12 This is a graph showing the results of DNA testing for a clinical sample of Mycobacterium tumefaciens;

[0060] Figure 13 This is a graph showing the detection results of cultured Mycobacterium chrysogenum DNA diluted to a concentration of 0.1 pg / μL;

[0061] Figure 14 This is a graph showing the DNA test results of a clinical sample of Mycobacterium Gordon.

[0062] Figure 15 This is a graph showing the detection results of cultured Mycobacterium Gordonii DNA diluted to a concentration of 0.1 pg / μL;

[0063] Figure 16 This is a diagram showing the results of DNA testing on a clinical sample of Mycobacterium bufo.

[0064] Figure 17 This is a graph showing the detection results of cultured Mycobacterium bufo diluted to a concentration of 0.1 pg / μL;

[0065] Figure 18 This is a graph showing the DNA test results of a clinical sample of Mycobacterium Malmo.

[0066] Figure 19 This is a graph showing the detection results of cultured Mycobacterium Malmos DNA diluted to a concentration of 0.1 pg / μL;

[0067] Figure 20 This is a diagram showing the DNA test results of a clinical sample of Mycobacterium Yongchuanense.

[0068] Figure 21 This is a graph showing the results of DNA testing in a clinical sample of Mycobacterium intermittentis;

[0069] Figure 22This is a diagram showing the results of DNA testing for Mycobacterium lymphoma in a clinical sample.

[0070] Figure 23 This is a diagram showing the DNA test results of a clinical sample of Mycobacterium kusmotoii.

[0071] Figure 24 This is a graph showing the results of E. coli DNA testing;

[0072] Figure 25 This is a graph showing the test results for purified water. Detailed Implementation

[0073] To further understand the present invention, preferred embodiments of the present invention are described below in conjunction with examples. However, it should be understood that these descriptions are only for further illustrating the features and advantages of the present invention, and are not intended to limit the present invention.

[0074] Table 1: Sequence Information

[0075]

[0076] The above sequences were synthesized by Shanghai Sangon Biotech. 9°N™ DNA Ligase and Buffer were purchased from NEWENGLAND Biolabs. The hybridization kit (LO-NA hyb) and probe fixation kit (LO-NA fix) were purchased from Shandong Lukang Orion Biotechnology. 2×Sapphire Amp Fast PCR Master Mix was purchased from TaKaRa.

[0077] DNA from clinical samples of tuberculous and non-tuberculous mycobacteria with known test results was analyzed. Clinical sample types included Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium kansasense, Mycobacterium abscessum, Mycobacterium intracellulare, Mycobacterium chrysogenum, Mycobacterium Gordonum, Mycobacterium bufotae, and Mycobacterium Malmosii. Simultaneously, DNA from clinical samples of Mycobacterium yongchuanensis, Mycobacterium intermittentum, Mycobacterium lymphadenum, and Mycobacterium kumamotoi, as well as purified water and Escherichia coli DNA, were introduced for cross-reactivity specificity verification. DNA was extracted from cultured Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium kansasense, Mycobacterium abscessum, Mycobacterium intracellulare, Mycobacterium chrysogenum, Mycobacterium Gordonum, Mycobacterium bufotae, and Mycobacterium Malmosii, and diluted to 0.1 pg / μL to verify detection sensitivity. All samples were obtained from the Guangxi Zhuang Autonomous Region Chest Hospital.

[0078] Example:

[0079] A method for detecting tuberculous and nontuberculous mycobacteria, comprising:

[0080] 1. Membrane chip fabrication:

[0081] The target probe and hybridization probe were diluted to 50 μM and fixed according to the probe immobilization kit instructions to obtain the membrane chip.

[0082] Table 2: The membrane chip arrangement of target probes and hybridization probes is as follows:

[0083]

[0084] 2. Preparation of probe mixture:

[0085] Dilute all upstream and downstream probes to a concentration of 1 μM, take 1.2 μL of each and mix them together, then add 600 μL of 1×TE solution to obtain the probe mixture; take 1.5 µL of the probe mixture, 5 µL of 1×TE solution, and 1.5 µL of the DNA from the above sample and mix them together in a PCR tube.

[0086] Table 3: Preparation of Probe Mixture

[0087]

[0088] 3. Denaturation, hybridization, and ligation reactions:

[0089] Set the PCR instrument program and run the above PCR tubes through the denaturation and hybridization reaction program (i.e., stages 1, 2, and 3) to obtain the hybridization product;

[0090] Table 4: Denaturation, Hybridization, and Ligation Reactions

[0091]

[0092] Table 5: Prepare the connecting mixture according to the following table:

[0093]

[0094] Add 2 µL of hybridization product to the corresponding PCR tube, cap the tube, place the PCR tube in the PCR instrument, and run the ligation reaction program (i.e., stages 4 and 5) to obtain the ligation product.

[0095] Table 6: Connection Reaction Program Parameters

[0096]

[0097] 4. PCR amplification:

[0098] Dilute the upstream and downstream primers to a concentration of 10 μM and prepare the PCR reaction system according to the formula in Table 7 to prepare the corresponding number of experiments.

[0099] Table 7: PCR reaction system

[0100]

[0101] Table 8: The PCR instrument program settings are as follows:

[0102]

[0103] 5. Membrane chip testing:

[0104] Table 9. Follow the instructions in the hybridization kit manual:

[0105]

[0106] 6. Test Results:

[0107] (1) Mycobacterium tuberculosis: Figure 2 For clinical sample DNA, obvious spots were detected at the locations of mycobacterial target probes, tuberculosis target probes, internal reference gene target probes, and hybridization probes, while no spots were detected at other locations; Figure 3 The concentration was 0.1 pg / μL. Spots were detected at the sites of the Mycobacterium spp. target probe, Mycobacterium tuberculosis target probe, and hybridization probe, but no spots were detected at other sites.

[0108] (2) Mycobacterium avium: Figure 4 For clinical sample DNA, obvious spots were detected at the locations of Mycobacterium spp. target probe, Mycobacterium avium target probe, internal reference gene target probe, and hybridization probe, while no spots were detected at other locations; Figure 5 The concentration was 0.1 pg / μL. Spots were detected at the sites of the Mycobacterium spp. target probe, Mycobacterium avium target probe, and hybridization probe, but no spots were detected at other sites.

[0109] (3) Mycobacterium kansasense: Figure 6 For clinical sample DNA, obvious spots were detected at the locations of Mycobacterium spp. target probe, Mycobacterium kansas target probe, internal reference gene target probe, and hybridization probe, while no spots were detected at other locations; Figure 7 The concentration was 0.1 pg / μL. Spots were detected at the target probe sites of Mycobacterium spp., Mycobacterium kansas, and hybridization probe, but no spots were detected at other sites.

[0110] (4) Mycobacterium abscessus: Figure 8 For clinical sample DNA, obvious spots were detected at the locations of Mycobacterium spp. target probe, Mycobacterium abscessus target probe, internal reference gene target probe, and hybridization probe, while no spots were detected at other locations; Figure 9 The concentration was 0.1 pg / μL. Spots were detected at the target probe sites of Mycobacterium spp., Mycobacterium abscessus, and hybridization probe, but no spots were detected at other sites.

[0111] (5) Intracellular mycobacteria: Figure 10 For clinical sample DNA, obvious spots were detected at the locations of mycobacterial target probes, intracellular mycobacterial target probes, internal reference gene target probes, and hybridization probes, while no spots were detected at other locations; Figure 11 The concentration was 0.1 pg / μL. Spots were detected at the sites of mycobacterial target probes, intracellular mycobacterial target probes, and hybridization probes, but no spots were found at other sites.

[0112] (6) Occasionally occurring mycobacteria: Figure 12 For clinical sample DNA, obvious spots were detected at the locations of Mycobacterium spp. target probe, random Mycobacterium target probe, internal reference gene target probe, and hybridization probe, while no spots were detected at other locations; Figure 13 The concentration was 0.1 pg / μL. Spots were detected at the sites of the Mycobacterium spp. target probe, the occasional Mycobacterium target probe, and the hybridization probe, but no spots were detected at other sites.

[0113] (7) Mycobacterium Gordon: Figure 14 For clinical sample DNA, obvious spots were detected at the locations of Mycobacterium spp. target probe, Mycobacterium Gordonii target probe, internal reference gene target probe, and hybridization probe, while no spots were detected at other locations; Figure 15 The concentration was 0.1 pg / μL. Spots were detected at the sites of the Mycobacterium spp. target probe, Mycobacterium Gordon's target probe, and hybridization probe, but no spots were detected at other sites.

[0114] (8) Mycobacterium bufotatum: Figure 16 For clinical sample DNA, obvious spots were detected at the locations of Mycobacterium spp. target probes, Mycobacterium bufota target probes, internal reference gene target probes, and hybridization probes, while no spots were detected at other locations; Figure 17 The concentration was 0.1 pg / μL. Spots were detected at the sites of the Mycobacterium spp. target probe, Mycobacterium bufo target probe, and hybridization probe, but no spots were detected at other sites.

[0115] (9) Mycobacterium Malmo: Figure 18 For clinical sample DNA, obvious spots were detected at the locations of Mycobacterium spp. target probe, Mycobacterium marmoset target probe, internal reference gene target probe, and hybridization probe, while no spots were detected at other locations; Figure 19 The concentration was 0.1 pg / μL. Spots were detected at the target probe sites of Mycobacterium spp., Mycobacterium Malmo, and hybridization probe, but no spots were detected at other sites.

[0116] (10) Mycobacterium yongchuanense: Figure 20For clinical sample DNA, obvious spots were detected at the locations of Mycobacterium spp. target probe, Mycobacterium yongchuanensis target probe, internal reference gene target probe, and hybridization probe, while no spots were detected at other locations;

[0117] (11) Intermittent Mycobacteria: Figure 21 For clinical sample DNA, obvious spots were detected at the locations of Mycobacterium spp. target probe, intermittent Mycobacterium target probe, internal reference gene target probe, and hybridization probe, while no spots were detected at other locations;

[0118] (12) Lymph node mycobacteria: Figure 22 For clinical sample DNA, obvious spots were detected at the locations of mycobacterial target probes, lymph node mycobacterial target probes, internal reference gene target probes, and hybridization probes, while no spots were detected at other locations;

[0119] (13) Kumamoto Mycobacterium: Figure 23 For clinical sample DNA, obvious spots were detected at the locations of Mycobacterium spp. target probe, Mycobacterium kusmotoii target probe, internal reference gene target probe, and hybridization probe, while no spots were detected at other locations;

[0120] (14) Escherichia coli: Figure 24 The DNA was from E. coli. Only the hybridization probe site showed obvious spots, while no spots appeared at other sites.

[0121] (15) Purified water: Figure 25 To purify the water, only the hybridization probe site showed obvious spots, while no spots appeared at other locations.

[0122] As shown in the image above:

[0123] 1. The DNA of clinical samples of Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium kansas, Mycobacterium abscessus, Mycobacterium intracellulare, Mycobacterium chrysogenum, Mycobacterium Gordon, Mycobacterium bufotae, and Mycobacterium Malmo were all correctly detected, and there was no cross-reactivity between them. At the same time, there was no cross-reactivity with the DNA of clinical samples of Mycobacterium yongchuanensis, Mycobacterium intermittentum, Mycobacterium lymphadenum, Mycobacterium kumamotoi, purified water, and Escherichia coli, which showed good specificity.

[0124] 2. Under low template concentration of 0.1 pg / μL, Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium kansas, Mycobacterium abscessus, Mycobacterium intracellularis, Mycobacterium chrysogenum, Mycobacterium gardenii, Mycobacterium bufo, and Mycobacterium marmosetum can all be clearly detected, with high sensitivity.

[0125] 3. The above description shows that the detection method of the present invention can quickly achieve multiplex detection of tuberculous and non-tuberculous mycobacteria such as Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium kansas, Mycobacterium abscessus, Mycobacterium intracellularis, Mycobacterium chrysogenum, Mycobacterium Gordonum, Mycobacterium bufota, and Mycobacterium Malmo, with good specificity and high sensitivity.

[0126] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, any non-essential improvements and changes made without departing from the inventive concept of the present invention are within the protection scope of the present invention.

Claims

1. A method for detecting tuberculous and non-tuberculous mycobacteria and its application, characterized in that: Ligases catalyze the formation of phosphodiester bonds between the 5' phosphate end and the 3' hydroxyl end of two adjacent DNA or RNA strands. These two DNA or RNA strands need to hybridize and accurately pair with complementary DNA or RNA strands without gaps, enabling the detection of each detection target. The ligases mentioned include, but are not limited to, commercially available Taq DNA Ligase, Ligase-65, 9°N DNA Ligase, HiFi Taq DNA Ligase, etc.

2. The method for detecting tuberculous and non-tuberculous mycobacteria according to claim 1 and its application, characterized in that: It consists of an upstream probe, a downstream probe, an upstream primer, a downstream primer, a complementary region of the upstream primer, an upstream linker, a downstream linker, a complementary region of the downstream primer, and a target probe, enabling the detection of each target. The upstream probe is a synthetically produced single-stranded DNA, single-stranded RNA, or single-stranded RNA-DNA sequence, with a length between 30 nt and 80 nt, consisting of an upstream primer complementary region and an upstream linker region. The upstream linker region is between 20 nt and 45 nt in length and is complementary to the template sequence. The downstream probe is a synthetically produced single-stranded DNA, single-stranded RNA, or single-stranded DNA-RNA sequence, with a length between 30 nt and 80 nt. The 5' end of the sequence is modified with a phosphate group, and it consists of a downstream linker region and a downstream primer complementary region. The downstream linker region is between 20 nt and 45 nt in length and is complementary to the template sequence. The upstream primer is a synthetically produced single-stranded DNA, single-stranded RNA, or single-stranded DNA-RNA with a length between 15 nt and 55 nt, and its sequence is completely or partially identical to the complementary region of the upstream primer. The downstream primer is a synthetic single-stranded DNA, single-stranded RNA, or single-stranded DNA-RNA with a length between 15 nt and 55 nt. The 5' end of the sequence is modified with biotin or digoxigenin groups, and the sequence is completely or partially complementary to the complementary region of the downstream primer.

3. Each detection target point according to claims 1 and 2 has a unique target probe that is the same as or complementary to the connection sequence of the upstream connection region + downstream connection region.

4. For each detection target according to claims 1, 2 and 3, upstream and downstream primers with the same or different sequences can be selected.

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