A primer set for differentiating Mycobacterium tuberculosis complex and nontuberculous mycobacteria and its application

Abstract

The present invention provides a primer set for differentiating Mycobacterium tuberculosis complex and non-tuberculous mycobacteria and its application. The primer set includes the nucleotide sequences shown in SEQ ID NO: 1-118. The present invention collects and analyzes the identification genes of Mycobacterium tuberculosis complex and non-tuberculous mycobacteria from the database, designs specific primers for the identification genes, combines multiplex PCR technology and targeted high-throughput sequencing technology, improves the detection sensitivity by ultra-multiplex PCR amplification and enrichment of the target, and improves the detection specificity based on the sequence reading of the gene sequencing platform, so as to achieve accurate and sensitive differentiation of mycobacteria. It has low detection cost, short detection cycle, high sensitivity, good specificity, and can effectively identify mixed infections, and has important application value in the detection of tuberculosis / NTM diseases.

Description

Technical Field

[0001] The present invention belongs to the technical field of gene detection, and particularly relates to a primer set for differentiating Mycobacterium tuberculosis complex and non-tuberculous Mycobacteria and its application. Background Art

[0002] The family Mycobacteriaceae belongs to the phylum Actinobacteria and contains a total of 5 genera, namely Mycobacterium, Mycolicibacter, Mycolicibacillus, Mycobacteroides, and Mycolicibacterium. Mycobacteria are irregularly shaped Gram-positive bacilli that are non-motile, usually acid-fast, and have no aerial hyphae or spores.

[0003] Non-tuberculous Mycobacteria (NTM) are a type of Mycobacteria other than Mycobacterium tuberculosis complex (MTBC) and Mycobacterium leprae. NTM is an environmental growing bacterium, and more than 190 species have been identified so far, and more than 40 species are pathogenic to humans. Clinically, NTM infections are mostly concurrent infections in patients with bronchiectasis and chronic obstructive pulmonary disease. Moreover, the clinical symptoms and X-ray manifestations of NTM lung disease are similar to those of pulmonary tuberculosis, but the treatment regimens are significantly different, and it is easy to misdiagnose and mistreat clinically, resulting in a protracted course for patients. With the continuous improvement of molecular diagnostic techniques and the popularization of disciplinary cognition, the incidence of NTM disease has been increasing year by year worldwide.

[0004] Mycobacterium tuberculosis and non-tuberculous Mycobacteria are difficult to differentiate in terms of morphology and acid-fast staining in pictures, and there are significant differences in their clinical treatments. Many non-tuberculous Mycobacteria are naturally resistant to anti-tuberculosis drugs, and the effect of conventional chemotherapy is not good. Therefore, the current identification of Mycobacteria is of great significance for the diagnosis, differential diagnosis, and effective chemotherapy of tuberculosis. Correct and rapid identification of Mycobacteria can avoid misdiagnosis, missed diagnosis, over-diagnosis, achieve individualized treatment, and is conducive to controlling the spread of tuberculosis and NTM disease.

[0005] Most of the current Mycobacteria identification methods are based on multiplex PCR techniques, including real-time fluorescence quantitative PCR technique, isothermal amplification technique, probe-reverse hybridization technique, and probe-melting curve technique, etc., which are all relatively convenient and fast. However, these reagents mostly identify bacterial species based on a specific SNP locus, the number of bacterial species that can be identified is limited, and it is also difficult to achieve subspecies identification.

[0006] The current first-generation sequencing-based mycobacterium species identification methods commonly used in clinical practice are mostly based on the 16S rRNA region. However, the resolution of the 16S rRNA region is insufficient, and some closely related species cannot be accurately identified. Therefore, developing a highly efficient and accurate methodology and primer set for mycobacterium identification has important application value in the detection of tuberculosis / NTM diseases. Summary of the Invention

[0007] Aiming at the deficiencies of the existing technology, the purpose of the present invention is to provide a primer set for differentiating Mycobacterium tuberculosis complex and nontuberculous mycobacteria and its application. The present invention combines multiplex PCR technology and targeted next-generation sequencing (tNGS). By enriching the target through ultra-multiplex PCR amplification, the detection sensitivity is improved. Based on the sequence reading of the gene sequencing platform (Illumina), the detection specificity is enhanced, thereby realizing the accurate and sensitive identification of mycobacteria. It has low detection cost, short detection cycle, high sensitivity, good specificity, and can effectively identify mixed infections.

[0008] To achieve the object of this invention, the present invention adopts the following technical solutions:

[0009] In the first aspect, the present invention provides a primer set for differentiating Mycobacterium tuberculosis complex and nontuberculous mycobacteria, and the primer set includes the nucleotide sequences shown in SEQ ID NO: 1-118.

[0010] Preferably, the primer set further includes primers for specifically amplifying an internal reference gene.

[0011] Preferably, the internal reference gene is the human zinc finger protein gene.

[0012] Preferably, the primers for specifically amplifying the internal reference gene include the nucleotide sequences shown in SEQ ID NO: 119-120.

[0013] Preferably, a common sequence is also linked to each primer sequence in the primer set.

[0014] Preferably, the common sequence includes the nucleotide sequence shown in SEQ ID NO: 121.

[0015] In the present invention, by consulting literature and public databases, the genome database of mycobacteria and common identification genes thereof are collected, the common identification genes are such as IS6110, IS1081, mtp64 or rpoB of the Mycobacterium tuberculosis complex, and 16S rRNA, rpoB, hsp65 or ITS of non-tuberculous mycobacteria, after the identification genes are found, specific primer sequences are designed for the identification genes, and internal reference specific primer sequences are designed at the same time, so as to obtain a primer set for distinguishing the Mycobacterium tuberculosis complex and non-tuberculous mycobacteria.

[0016] During the strain identification process, there are certain differences in the sequences of different strains of the same bacterial species. The signature SNP (a single site or a certain region or several regions) exists in different strains of the bacterial species or different strains of closely related species. A pair of primer sequences can amplify multiple bacterial species at the same time, among which some bacterial species can be identified by signature SNPs; multiple pairs of primers combined can achieve accurate identification of bacterial species.

[0017] In a second aspect, the present invention provides a kit for identifying Mycobacterium tuberculosis complex and non-tuberculous mycobacteria, wherein the kit comprises the primer set for identifying Mycobacterium tuberculosis complex and non-tuberculous mycobacteria according to the first aspect.

[0018] Preferably, the kit further comprises a sequencing adapter and a blocking primer.

[0019] In the present invention, the 5' end of each specific primer sequence is connected to a common sequence (5'-gactgccgctggttggatg-3', SEQ ID NO: 121) as a PCR primer sequence, and the function of the common sequence is to connect the sequencing adapter and each specific primer. The 3' end of the sequencing adapter of the Illumina platform is connected to the common sequence as a sequencing adapter primer, and the function of the common sequence is to connect the PCR primer and amplify the target region.

[0020] Preferably, the blocking primer comprises the nucleotide sequence shown in SEQ ID NO:122.

[0021] In the present invention, the reverse complementary sequence of the common sequence is used as a blocking primer (5'-catccaaccagcggcagtc-3', SEQ ID NO: 122) to avoid the connection between the adapter primer and the specific primer during the process of amplifying the sample target region by PCR primer sequence.

[0022] Preferably, the sequencing adapter is also connected to a common sequence.

[0023] In a third aspect, the present invention provides a device for distinguishing Mycobacterium tuberculosis complex from non-tuberculous mycobacteria, the device comprising:

[0024] Library construction unit: Using the kit for differentiating Mycobacterium tuberculosis complex and non-tuberculous mycobacteria described in the second aspect to prepare a library construction primer system, performing ultra-multiplex PCR amplification on the template DNA to obtain an ultra-multiplex PCR library;

[0025] Sequencing unit: Purifying and performing high-throughput sequencing on the ultra-multiplex PCR library;

[0026] Analysis unit: Differentiating Mycobacterium tuberculosis complex and non-tuberculous mycobacteria according to the high-throughput sequencing results.

[0027] Preferably, the library construction primer system includes: primer sets, sequencing adapters, and blocking primers.

[0028] Preferably, the primer sets, sequencing adapters, and blocking primers in the library construction primer system are mixed in a ratio of (4 - 5):(2 - 2.5):(0.8 - 1.2), and the ratios can be, for example, 4:2:0.8, 4:2.5:0.8, 4:2.5:1.2, 5:2:0.8, 5:2.5:0.8, or 5:2.5:1.2, etc.

[0029] Preferably, the structure of the ultra-multiplex PCR library is Illumina sequencing adapter - common sequence - target region - common sequence - Illumina sequencing adapter.

[0030] In the present invention, a one-step method is used to construct the ultra-multiplex PCR library. The conventional PCR library construction steps on the Illumina platform are to perform nucleic acid amplification using primer sets, and then perform adapter ligation after the amplification products are not repaired. Two rounds of PCR amplification are required, which is generally verbally referred to as a two-step library construction method; our one-step library construction method performs adapter ligation while amplifying, and only one round of PCR amplification is required, so it is called a one-step library construction method. The entire library construction process does not require opening the lid, avoiding contamination; and saving manual operation.

[0031] Fourth aspect, the present invention provides a method for differentiating Mycobacterium tuberculosis complex and non-tuberculous mycobacteria for non-diagnostic or non-therapeutic purposes, the method comprising:

[0032] Using the kit for differentiating Mycobacterium tuberculosis complex and non-tuberculous mycobacteria described in the second aspect to prepare a library construction primer system, performing ultra-multiplex PCR amplification on the template DNA to obtain an ultra-multiplex PCR library; purifying and performing high-throughput sequencing on the ultra-multiplex PCR library; differentiating Mycobacterium tuberculosis complex and non-tuberculous mycobacteria according to the high-throughput sequencing results.

[0033] Preferably, the library construction primer system includes: primer sets, sequencing adapters, and blocking primers.

[0034] Preferably, in the library construction primer system, the primer set, sequencing adapter, and blocking primer are mixed in a ratio of (4-5):(2-2.5):(0.8-1.2). The ratio can be, for example, 4:2:0.8, 4:2.5:0.8, 4:2.5:1.2, 5:2:0.8, 5:2.5:0.8, or 5:2.5:1.2, etc.

[0035] Preferably, the structure of the ultra-multiplex PCR library is Illumina sequencing adapter - common sequence - target region - common sequence - Illumina sequencing adapter.

[0036] In a fifth aspect, the present invention provides the use of the primer set for differentiating Mycobacterium tuberculosis complex and non-tuberculous mycobacteria described in the first aspect and / or the kit for differentiating Mycobacterium tuberculosis complex and non-tuberculous mycobacteria described in the second aspect in the preparation of tuberculosis / NTM disease detection products.

[0037] The numerical ranges described in the present invention not only include the above-listed point values, but also any point values between the above numerical ranges not listed. Due to space limitations and for the sake of brevity, the present invention does not exhaustively list the specific point values included in the described ranges.

[0038] Compared with the prior art, the present invention has the following beneficial effects:

[0039] (1) The present invention combines multiplex PCR technology and targeted high-throughput sequencing technology (Targeted next-generation sequencing, tNGS), improves the detection sensitivity by ultra-multiplex PCR amplification and enrichment of the target, and improves the detection specificity based on sequence reading of the gene sequencing platform (Illumina), thereby achieving accurate and sensitive identification of mycobacteria. It has low detection cost, short detection cycle, high sensitivity, good specificity, and can effectively identify mixed infections.

[0040] (2) The primer set and kit described in the present invention show high sensitivity in the identification of mycobacteria, can stably detect at least 40 CFU / mL of Mycobacterium tuberculosis, can stably detect 65 - 73 CFU / mL of non-tuberculous mycobacteria other than Mycobacterium terrae, and can stably detect 118.9 CFU / mL of Mycobacterium terrae. In addition, the primer set or kit can accurately distinguish between mixed infections of Mycobacterium tuberculosis complex and non-tuberculous mycobacteria, and has important application value in the detection of tuberculosis / NTM diseases.

[0041] (3) The identification method of the present invention can also prevent aerosol contamination within the batch during the detection process. In the conventional PCR process, the amplification product is first amplified and then transferred for the second round of PCR to connect the sequencing adapter. During the transfer process, there is a possibility of aerosol contamination between the first round of PCR products between samples; in contrast, the identification method of the present invention can prevent aerosol contamination within the batch. After the template is added to the reaction system, amplification and adapter connection are performed simultaneously in the same reaction system. After the PCR is completed, the library has been connected to the corresponding sequencing adapter, avoiding potential contamination during the transfer process. BRIEF DESCRIPTION OF THE DRAWINGS

[0042] Figure 1 These are the results of determination of different concentrations of reference substances of Mycobacterium tuberculosis.

[0043] Figure 2 The result is the 95% detection limit of Mycobacterium tuberculosis.

[0044] Figure 3 The results are 95% detection limit for 4 types of nontuberculous mycobacteria. DETAILED DESCRIPTION

[0045] The technical solution of the present invention is further described below by specific implementation methods. It should be understood by those skilled in the art that the embodiments are only to help understand the present invention and should not be regarded as specific limitations of the present invention.

[0046] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0047] Example 1 A primer set for distinguishing Mycobacterium tuberculosis complex and non-tuberculous mycobacteria

[0048] This embodiment provides a primer set for identifying Mycobacterium tuberculosis complex and non-tuberculous mycobacteria, and the primer sequences are shown in Table 1. In the primer set, a pair of primer sequences can simultaneously amplify multiple bacterial species, some of which can be identified by marker SNPs; multiple pairs of primers can be combined to achieve accurate identification of bacterial species.

[0049] Table 1

[0050]

[0051]

[0052]

[0053]

[0054]

[0055] Example 2 A kit for differentiating Mycobacterium tuberculosis complex and non-tuberculous mycobacteria

[0056] This example provides a kit for differentiating Mycobacterium tuberculosis complex and non-tuberculous mycobacteria. The kit includes the primer set for differentiating Mycobacterium tuberculosis complex and non-tuberculous mycobacteria in Example 1. The kit also includes the reagents for one-step library construction, and the reagents include: sequencing adapters, blocking primers, DNA polymerase, and amplification buffer.

[0057] Example 3 Mycobacterium differentiation

[0058] The kit in Example 2 was used to measure gradient dilution samples of Mycobacterium tuberculosis and several non-tuberculous mycobacteria (NTM) to evaluate its detection performance.

[0059] Among them, the Mycobacterium tuberculosis samples to be tested were the lowest detection limit reference products (S1, 1×10 3 colony-forming units / mL, S2, 1×10 2 colony-forming units / mL, S3, 1×10 1 colony-forming units / mL, S4, 1×1 0 colony-forming units / mL) in the Mycobacterium tuberculosis PCR detection kit national reference product (purchased from the National Institutes for Food and Drug Control, product number 230030).

[0060] The NTM samples to be tested were selected as follows: (1) Mycobacterium smegmatis, Mycobacterium abscessus, Mycobacterium terrae (purchased from the China General Microbiological Culture Collection Center); (2) Mycobacterium neoaurum (purchased from the Guangdong Culture Collection Center).

[0061] (1) Stability assessment: Using reference products S1, S2, S3, and S4 as materials, after nucleic acid extraction, library construction and sequencing were performed with 20 technical replicates.

[0062] The library construction steps are as follows: Prepare a one-step library construction primer system, and the components and their concentrations in the system are shown in Table 2.

[0063] Table 2

[0064] Component Concentration Enzyme 1 U / μL Primer 20 pmol / μL Buffer 2× Buffer Template 40 ng / μL

[0065] Perform ultra-multiplex PCR amplification on the template DNA for PCR amplification, and the PCR amplification program is set as shown in Table 3.

[0066] Table 3

[0067]

[0068]

[0069] After amplification, a super-multiplex PCR library is obtained, and finally the library structure is "Illumina sequencing adapter - common sequence - target region - common sequence - Illumina sequencing adapter". Among them, different template DNAs use different sequencing adapters.

[0070] Each super-multiplex PCR library is purified, and after purification, the libraries are mixed to obtain a library for Mycobacterium identification.

[0071] The super-multiplex PCR library is subjected to high-throughput sequencing, and bioinformatics analysis methods are used to analyze the sequencing data, and finally the Mycobacterium identification results are obtained.

[0072] The measurement results are as Figure 1 and Table 4, Figure 1 which are the measurement results of different concentration reference products of Mycobacterium tuberculosis. It can be seen from the figure that all reference products except S1 with the lowest concentration are detected at 100%, while only 6 samples of S1 are detected (positive detection rate 30%). At the same time, as the concentration of S1 increases, the coefficient of variation (CV) of S1, S2, S3, and S4 shows a gradually decreasing trend. The CVs of S3 and S4 are 20.37% and 18.52% respectively, and show relatively stable detection performance at a concentration of 1×10 3 CFU / mL and above. Table 4 is the stability evaluation result based on the Mycobacterium tuberculosis reference product.

[0073] Table 4

[0074]

[0075] (2) Sensitivity evaluation: Further evaluate the detection sensitivity of the kit. Using Mycobacterium tuberculosis and the above 4 non-tuberculous mycobacteria as research materials, they are diluted to corresponding concentration gradients with normal saline as shown in Table 5. After library construction and sequencing, the LoD 95％ (95% detection limit) of each bacterium is calculated by the observation method and the Probit analysis method. Finally, the LoD 95％ of Mycobacterium tuberculosis measured by the observation method is 95％ 40.0 CFU / mL, while the LoD Figure 2 inferred by the fitting method is 30.8 CFU / mL ( Figure 2 , Table 5).

[0076] Table 5

[0077] Strain Concentration gradient Technical replication <![CDATA[LoD 95％ > Mycobacterium tuberculosis 1,2,4,8,10,20,40,80,100,200,400,800,1000 20 30.8 / 40.0 Mycobacterium smegmatis 1,5,10,50,100,500,1000 5 72.5 Mycobacterium abscessus 1,5,10,50,100,500,1000 5 72.0 Mycobacterium terrae 1,5,10,50,100,500,1000 5 118.9 Mycobacterium neoaurum 1,5,10,50,100,500,1000 5 65.7

[0078] Figure 3 The determination results of the 95% detection limits of 4 non-tuberculous mycobacteria. The LoDs of Mycobacterium smegmatis, Mycobacterium abscessus, Mycobacterium terrae, and Mycobacterium neoaurum inferred by the fitting method 95 were 72.5 CFU / mL, 72.0 CFU / mL, 118.9 CFU / mL, and 65.7 CFU / mL respectively. It can be seen from this that this kit can stably detect Mycobacterium tuberculosis at least at 40 CFU / mL. Non-tuberculous mycobacteria other than Mycobacterium terrae can be stably detected at 65 - 73 CFU / mL, and Mycobacterium terrae can be stably detected at 118.9 CFU / mL. This kit generally shows high sensitivity.

[0079] Example 4 Identification of Mycobacterium mixed infections

[0080] Use the kit in Example 2 to measure the proportionally gradient mixed samples to evaluate its detection performance for mixed infections. The library construction steps refer to Example 3.

[0081] Among them, the Mycobacterium tuberculosis samples to be tested use the positive reference products P1 and P2 (1×10 3 CFU / mL) in the national reference product (purchased from the National Institutes for Food and Drug Control) of the Mycobacterium tuberculosis PCR detection kit. The NTM strains to be tested are Mycobacterium abscessus and Mycobacterium terrae (purchased from the China General Microbiological Culture Collection Center).

[0082] Gradient dilute the NTM strains to be tested with normal saline. The Mycobacterium tuberculosis complex reference product and Mycobacterium abscessus are respectively mixed at ratios of (1×10 3 CFU / mL : 1×10 2 CFU / mL, 1×10 3 CFU / mL : 1×10 3 CFU / mL, 1×10 3 CFU / mL : 1×10 4 CFU / mL, 1×10 3 CFU / mL : 1×10 5 CFU / mL, 1×10 3 CFU / mL : 1×10 6 CFU / mL) to simulate different degrees of mixed infections of tuberculosis and non-tuberculosis.

[0083] Gradient dilute the NTM strains to be tested with normal saline. Mycobacterium abscessus and Mycobacterium terrae are respectively mixed at ratios of (1×10 6 CFU / mL : 1×10 3 CFU / mL, 1×10 5 CFU / mL : 1×103 CFU / mL, 1×10 4 CFU / mL: 1×10 3 CFU / mL, 1×10 3 CFU / mL: 1×10 3 CFU / mL, 1×10 2 CFU / mL: 1×10 3 CFU / mL, 1×10 3 CFU / mL: 1×10 2 CFU / mL, 1×10 3 CFU / mL: 1×10 4 CFU / mL, 1×10 3 CFU / mL: 1×10 5 CFU / mL, 1×10 3 CFU / mL: 1×10 6 CFU / mL) were mixed in the ratio of to simulate different non - tuberculosis mixed infections.

[0084] Nucleic acid extraction was performed on the mixed samples, and library construction and sequencing were performed on the extracted nucleic acids. The detection results of the simulated mixed samples are shown in Table 6.

[0085] Table 6

[0086]

[0087]

[0088] The results showed that this primer set could accurately distinguish the mixed infection of Mycobacterium tuberculosis complex and non - tuberculous mycobacteria; for the mixed infection of non - tuberculous mycobacteria, when the concentration of one bacterium was around the detection limit (such as 1×10 2 CFU / mL) and the concentration of the other bacterium was higher than 1×10 5 CFU / mL, the non - tuberculous mycobacteria with low load might not be detected; for the mixed infection of non - tuberculous mycobacteria with a concentration higher than 1×10 3 CFU / mL, the primer set could accurately distinguish.

[0089] In summary, the present invention provides a method and primer set for differentiating mycobacteria by one - step library construction and sequencing. The method improves the detection sensitivity through ultra - multiplex PCR amplification and enrichment of target sequences, and improves the detection specificity based on sequence reading of the gene sequencing platform, thereby realizing accurate and sensitive differentiation of mycobacteria. It has low detection cost, short detection cycle, high sensitivity, good specificity, and can effectively distinguish mixed infections, and has important application value in the detection of tuberculosis / NTM diseases.

[0090] The applicant declares that the above description is only a specific implementation manner of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention fall within the protection scope and the disclosure scope of the present invention.

Claims

1. A primer set for differentiating Mycobacterium tuberculosis complex and nontuberculous mycobacteria, characterized in that, The primer set includes nucleotide sequences shown in SEQ ID NO: 1-118; a common sequence is further connected to the 5'-end of each primer sequence in SEQ ID NO: 1-118; the common sequence includes the nucleotide sequence shown in SEQ ID NO: 121; The primer set further includes a sequencing adapter and a blocking primer; the blocking primer includes the nucleotide sequence shown in SEQ ID NO: 122; the 3'-end of the sequencing adapter is connected to the common sequence shown in SEQ ID NO: 121 as a sequencing adapter primer.

2. The primer set for differentiating Mycobacterium tuberculosis complex and non-tuberculous mycobacteria according to claim 1, characterized in that, The primer set further includes primers for specifically amplifying a reference gene; the reference gene is a human zinc finger protein gene.

3. The primer set for differentiating Mycobacterium tuberculosis complex and non-tuberculous mycobacteria according to claim 2, characterized in that, The primers for specifically amplifying the reference gene include the nucleotide sequences shown in SEQ ID NO: 119-120.

4. A kit for differentiating Mycobacterium tuberculosis complex and non-tuberculous mycobacteria, characterized in that, The kit includes the primer set for differentiating Mycobacterium tuberculosis complex and nontuberculous mycobacteria according to any one of claims 1-3.

5. An apparatus for differentiating Mycobacterium tuberculosis complex and nontuberculous mycobacteria, characterized in that, The device includes: A library construction unit: preparing a library construction primer system using the kit for differentiating Mycobacterium tuberculosis complex and nontuberculous mycobacteria according to claim 4, performing ultra-multiplex PCR amplification on template DNA to obtain an ultra-multiplex PCR library; A sequencing unit: purifying and performing high-throughput sequencing on the ultra-multiplex PCR library; An analysis unit: differentiating Mycobacterium tuberculosis complex and nontuberculous mycobacteria according to the high-throughput sequencing results.

6. The device for differentiating Mycobacterium tuberculosis from non-tuberculous mycobacteria according to claim 5, characterized in that, In the library construction primer system, the primer set, the sequencing adapter, and the blocking primer are mixed in a ratio of (4-5):(2-2.5):(0.8-1.2).

7. The device for differentiating Mycobacterium tuberculosis from non-tuberculous mycobacteria according to claim 5, characterized in that, The structure of the ultra-multiplex PCR library is Illumina sequencing adapter - common sequence - target region - common sequence - Illumina sequencing adapter.

8. A method for differentiating Mycobacterium tuberculosis complex and non-tuberculous mycobacteria for non-diagnostic or non-therapeutic purposes, characterized in that, The method includes: Preparing a library construction primer system using the kit for differentiating Mycobacterium tuberculosis complex and nontuberculous mycobacteria according to claim 4, performing ultra-multiplex PCR amplification on template DNA to obtain an ultra-multiplex PCR library; purifying and performing high-throughput sequencing on the ultra-multiplex PCR library; differentiating Mycobacterium tuberculosis complex and nontuberculous mycobacteria according to the high-throughput sequencing results.

9. The method for differentiating Mycobacterium tuberculosis complex and nontuberculous mycobacteria for non-diagnostic or non-therapeutic purposes according to claim 8, characterized in that, In the library construction primer system, the primer set, the sequencing adapter, and the blocking primer are mixed in a ratio of (4-5):(2-2.5):(0.8-1.2).

10. The method for differentiating Mycobacterium tuberculosis complex and nontuberculous mycobacteria for non-diagnostic or non-therapeutic purposes according to claim 8, wherein The structure of the ultra-multiplex PCR library is Illumina sequencing adapter - common sequence - target region - common sequence - Illumina sequencing adapter.

11. Use of the primer set for differentiating Mycobacterium tuberculosis complex and nontuberculous mycobacteria according to any one of claims 1-3 and / or the kit for differentiating Mycobacterium tuberculosis complex and nontuberculous mycobacteria according to claim 4 in the preparation of tuberculosis / NTM disease detection products.

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