Composition for confirming drug resistance of mycobacterium tuberculosis

Abstract

The invention belongs to the field of biological detection. In particular to a composition for sequencing detection, and more particularly relates to a composition for confirming the drug resistance of mycobacterium tuberculosis. By using the composition disclosed by the invention, 30 targets such as ahpC, atpE, ddn, eis, embB, erm, ethA, fabG1, fbiA, fbiB, fbiC, fgd1, gidB, gyrA, gyrB, inhA, katG, pepQ, pncA, rplC, rpoB, rpsA, rpsL, rrL, rrs, Rv0678, Rv1979c, Rv2983, tlyA and ubiA can be simultaneously amplified and enriched in a tube, and corresponding to the detection and confirmation of the drug resistance of 14 anti-tuberculosis drugs, the composition disclosed by the invention uses as few primer groups as possible to detect as many targets and the corresponding anti-tuberculosis drugs as possible.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection, and in particular, relates to a composition for sequencing detection, and more particularly, to a composition for confirming the drug resistance of Mycobacterium tuberculosis. Background Art

[0002] Tuberculosis (TB), caused by infection with Mycobacterium tuberculosis (MTB), is a major global disease that seriously endangers human health. According to the WHO's "2024 Global Tuberculosis Report", there will be 10.8 million new tuberculosis patients and about 1.2 million deaths from tuberculosis in 2023. About one-third of the world's population has been infected with MTB, and about 5-10% of people infected with Mycobacterium tuberculosis may develop active tuberculosis in their lifetime. Faced with the severe tuberculosis epidemic and the increasing number of HIV co-infections, timely identification of Mycobacterium tuberculosis infected people and providing appropriate treatment for patients are the key to controlling the spread of tuberculosis.

[0003] The multidrug resistance problem of Mycobacterium tuberculosis is becoming increasingly serious, and resistance to mainstream anti-tuberculosis drugs including isoniazid, rifampicin and ethambutol is increasing, making the disease more difficult to control. In 2023, there were 400,000 patients with multidrug-resistant / rifampicin-resistant tuberculosis, of which only about 44% of drug-resistant patients were diagnosed and included in standardized treatment. There is still a gap in the discovery and treatment of drug-resistant tuberculosis, which remains a public health crisis. In July 2023, WHO recommended the use of targeted high-throughput sequencing technology (tNGS) to detect drug-resistant tuberculosis, which can detect drug resistance of all drugs included in the anti-tuberculosis treatment regimen, improve the cure rate of drug-resistant tuberculosis, shorten the time for patients to excrete bacteria, and reduce the spread of the disease. It is worth mentioning that WHO also recommended three long amplicon tNGS products. Long amplicons have unique advantages over traditional short amplicons based on hotspot mutations. Long amplicons cover a wider range of drug-resistant genes and can detect non-hotspot mutations included in the WHO drug-resistant mutation catalog that are included in the first and second level drug-resistant mutations, as well as mutations that have not yet been characterized but may be related to drug resistance, making the detection of drug-resistant mutations more comprehensive; they can also discover new mutations that may appear in the covered gene range, allowing researchers to study them.

[0004] In addition, long amplification is also an inherent requirement for detecting pncA resistance mutations in Mycobacterium tuberculosis. The pyrazinamidase encoded by the pncA gene can activate the first-line anti-tuberculosis drug pyrazinamide (PZA) into pyrazinoic acid to exert its antibacterial effect. In drug resistance detection, mutations in the pncA gene are usually strongly correlated with PZA resistance. pncA resistance mutations are very densely arranged on the genome, which means that traditional short amplicon schemes may not be able to fully cover all mutation sites, so long amplicon design is needed to ensure that these mutations can be detected. Long amplicon PCR technology can cover larger DNA fragments, thereby improving the coverage and accuracy of detecting pncA resistance mutations.

[0005] Although the third-generation sequencing technology can detect long amplicons, the technology is not mature enough, lacks stability, and is relatively expensive. In contrast, the second-generation sequencing technology (Next-Generation Sequencing, NGS) has advantages in accuracy, cost-effectiveness, and user-friendliness. The second-generation sequencing can provide high-throughput, low-cost sequencing data, which is very useful for detecting the drug-resistant genotype of Mycobacterium tuberculosis, especially when multiple drug-resistant genes and multiple sites need to be detected.

[0006] Therefore, there is a need in the art for a composition that can detect multiple drug-resistant genes and sites and is easy to operate and accurate in detection. Summary of the invention

[0007] In view of this, in a first aspect, the present invention provides a composition for confirming the drug resistance of Mycobacterium tuberculosis, comprising a primer set for detecting at least four of the following targets:

[0008] A primer set for amplifying the ahpC gene as shown in SEQ ID NOs. 1 to 2;

[0009] A primer set for amplifying the atpE gene as shown in SEQ ID NOs. 3 to 4;

[0010] A primer set for amplifying the ddn gene as shown in SEQ ID NOs. 5 to 6;

[0011] A primer set for amplifying the eis gene as shown in SEQ ID NOs. 7 to 8;

[0012] A primer set for amplifying the embB gene as shown in SEQ ID NOs. 9 to 10;

[0013] A primer set for amplifying the erm gene as shown in SEQ ID NOs. 11 to 12;

[0014] A primer set for amplifying the ethA gene as shown in SEQ ID NOs. 13 to 16;

[0015] A primer set for amplifying the fabG1 gene as shown in SEQ ID NOs. 17 to 18;

[0016] A primer set for amplifying the fbiA gene as shown in SEQ ID NOs. 19 to 20;

[0017] A primer set for amplifying the fbiB gene as shown in SEQ ID NOs. 21 to 22;

[0018] A primer set for amplifying the fbiC gene as shown in SEQ ID NOs. 23 to 28;

[0019] A primer set for amplifying the fgd1 gene as shown in SEQ ID NOs. 29 to 30;

[0020] A primer set for amplifying the gidB gene as shown in SEQ ID NOs. 31 to 32;

[0021] A primer set for amplifying the gyrA gene as shown in SEQ ID NOs. 33 to 34;

[0022] A primer set for amplifying the gyrB gene as shown in SEQ ID NOs. 35 to 36;

[0023] A primer set for amplifying the inhA gene as shown in SEQ ID NOs. 37 to 38;

[0024] A primer set for amplifying the katG gene as shown in SEQ ID NOs. 39 to 42;

[0025] A primer set for amplifying the pepQ gene as shown in SEQ ID NOs. 43 to 46;

[0026] A primer set for amplifying the pncA gene as shown in SEQ ID NOs. 47 to 48;

[0027] A primer set for amplifying the rplC gene as shown in SEQ ID NOs. 49 to 50;

[0028] A primer set for amplifying the rpoB gene as shown in SEQ ID NOs. 51 to 52;

[0029] A primer set for amplifying the rpsA gene as shown in SEQ ID NOs. 53 to 56;

[0030] A primer set for amplifying the rpsL gene as shown in SEQ ID NOs. 57 to 58;

[0031] A primer set for amplifying the rrL gene as shown in SEQ ID NOs. 59 to 60;

[0032] A primer set for amplifying the rrs gene as shown in SEQ ID NOs. 61 to 62;

[0033] A primer set for amplifying the rv0678 gene as shown in SEQ ID NOs. 63-64;

[0034] A primer set for amplifying the Rv1979c gene as shown in SEQ ID NOs. 65 to 68;

[0035] A primer set for amplifying the Rv2983 gene as shown in SEQ ID NOs. 69 to 70;

[0036] A primer set for amplifying the tlyA gene as shown in SEQ ID NOs. 71 to 72; or

[0037] The primer set for amplifying the ubiA gene is shown in SEQ ID NOs. 73-74.

[0038] Furthermore, the present invention provides a composition for confirming the drug resistance of Mycobacterium tuberculosis, comprising at least 8 targets in the primer set shown above.

[0039] Furthermore, the present invention provides a composition for confirming the drug resistance of Mycobacterium tuberculosis, comprising at least 10 targets in the primer set as shown above.

[0040] Furthermore, the present invention provides a composition for confirming the drug resistance of Mycobacterium tuberculosis, comprising at least 20 targets in the primer set as shown above.

[0041] Furthermore, the present invention provides a composition for confirming drug resistance of Mycobacterium tuberculosis, comprising at least 25 targets in the primer set as shown above.

[0042] By using the composition of the present invention, multiple targets can be amplified and enriched simultaneously in one tube, and multiple targets related to tuberculosis drug resistance can be detected, thereby simplifying the analysis work and making the entire detection more accurate and efficient.

[0043] It should be noted that, those skilled in the art know that if 30 targets can be amplified by PCR in one tube at the same time, then there is no doubt that any combination of these 30 targets (for example, any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22 ... 30) can be amplified by PCR in one tube.

[0044] Furthermore, the present invention provides a composition for confirming the drug resistance of Mycobacterium tuberculosis, comprising:

[0045] A primer set for amplifying the ahpC gene as shown in SEQ ID NOs. 1 to 2;

[0046] A primer set for amplifying the atpE gene as shown in SEQ ID NOs. 3 to 4;

[0047] A primer set for amplifying the ddn gene as shown in SEQ ID NOs. 5 to 6;

[0048] A primer set for amplifying the eis gene as shown in SEQ ID NOs. 7 to 8;

[0049] A primer set for amplifying the embB gene as shown in SEQ ID NOs. 9 to 10;

[0050] A primer set for amplifying the erm gene as shown in SEQ ID NOs. 11 to 12;

[0051] A primer set for amplifying the ethA gene as shown in SEQ ID NOs. 13 to 16;

[0052] A primer set for amplifying the fabG1 gene as shown in SEQ ID NOs. 17 to 18;

[0053] A primer set for amplifying the fbiA gene as shown in SEQ ID NOs. 19 to 20;

[0054] A primer set for amplifying the fbiB gene as shown in SEQ ID NOs. 21 to 22;

[0055] A primer set for amplifying the fbiC gene as shown in SEQ ID NOs. 23 to 28;

[0056] A primer set for amplifying the fgd1 gene as shown in SEQ ID NOs. 29 to 30;

[0057] A primer set for amplifying the gidB gene as shown in SEQ ID NOs. 31 to 32;

[0058] A primer set for amplifying the gyrA gene as shown in SEQ ID NOs. 33 to 34;

[0059] A primer set for amplifying the gyrB gene as shown in SEQ ID NOs. 35 to 36;

[0060] A primer set for amplifying the inhA gene as shown in SEQ ID NOs. 37 to 38;

[0061] A primer set for amplifying the katG gene as shown in SEQ ID NOs. 39 to 42;

[0062] A primer set for amplifying the pepQ gene as shown in SEQ ID NOs. 43 to 46;

[0063] A primer set for amplifying the pncA gene as shown in SEQ ID NOs. 47 to 48;

[0064] A primer set for amplifying the rplC gene as shown in SEQ ID NOs. 49 to 50;

[0065] A primer set for amplifying the rpoB gene as shown in SEQ ID NOs. 51 to 52;

[0066] A primer set for amplifying the rpsA gene as shown in SEQ ID NOs. 53 to 56;

[0067] A primer set for amplifying the rpsL gene as shown in SEQ ID NOs. 57 to 58;

[0068] A primer set for amplifying the rrL gene as shown in SEQ ID NOs. 59 to 60;

[0069] A primer set for amplifying the rrs gene as shown in SEQ ID NOs. 61 to 62;

[0070] A primer set for amplifying the rv0678 gene as shown in SEQ ID NOs. 63-64;

[0071] A primer set for amplifying the Rv1979c gene as shown in SEQ ID NOs. 65 to 68;

[0072] A primer set for amplifying the Rv2983 gene as shown in SEQ ID NOs. 69 to 70;

[0073] A primer set for amplifying the tlyA gene as shown in SEQ ID NOs. 71 to 72; and

[0074] The primer set for amplifying the ubiA gene is shown in SEQ ID NOs. 73-74.

[0075] Using the above-mentioned composition of the present invention, 30 targets including ahpC, atpE, ddn, eis, embB, erm, ethA, fabG1, fbiA, fbiB, fbiC, fgd1, gidB, gyrA, gyrB, inhA, katG, pepQ, pncA, rplC, rpoB, rpsA, rpsL, rrL, rrs, rv0678, Rv1979c, Rv2983, tlyA, and ubiA can be amplified and enriched simultaneously in one tube, corresponding to the detection and confirmation of 14 anti-tuberculosis drugs. The composition of the present invention uses as few primer sets as possible to detect as many targets as possible and the corresponding anti-tuberculosis drugs.

[0076] Optionally, the primer set carries a linker sequence to facilitate sequencing.

[0077] Optionally, the primer set does not have a linker sequence.

[0078] In some specific embodiments, the components of the composition are present in the same package.

[0079] Furthermore, the components of the composition of the present invention are present in a mixed form.

[0080] In a second aspect, the present invention provides a use of the above composition for preparing a kit for confirming the drug resistance of Mycobacterium tuberculosis.

[0081] In a third aspect, the present invention provides a kit for confirming the drug resistance of Mycobacterium tuberculosis, comprising the above composition.

[0082] Furthermore, the kit also includes at least one of the following: a nucleic acid extraction reagent, a nucleic acid amplification reagent, a library construction reagent, and a sequencing reagent.

[0083] Furthermore, the nucleic acid extraction reagent may be a reagent for extracting DNA from samples such as alveolar lavage fluid and sputum.

[0084] Furthermore, the nucleic acid amplification reagent includes DNA polymerase, dNTP, buffer, and Mg 2+ .

[0085] Furthermore, the library construction reagent includes an enzyme. After amplification, the composition of the present invention is digested with an enzyme and then the library is constructed.

[0086] Furthermore, the library construction reagents include fragmentation reagents, end repair reagents, adapter ligation reagents, and library amplification reagents.

[0087] In a fourth aspect, the present invention provides a composition for use in preparing a reagent for confirming drug resistance of Mycobacterium tuberculosis, wherein the detection comprises:

[0088] 1) Extracting or releasing nucleic acid from the sample to be tested;

[0089] 2) amplifying using the composition as described above to obtain an amplified product;

[0090] 3) Processing the amplified products and building a library; and

[0091] 4) Sequencing and analyzing the results.

[0092] Furthermore, the amplification conditions are:

[0093]

[0094] BRIEF DESCRIPTION OF THE DRAWINGS

[0095] Figure 1 Construct a flow chart for the library;

[0096] Figure 2 is the depth map of the amplicon of the composition of the present invention;

[0097] Figure 3 This is the depth map of the pncA gene amplicon. DETAILED DESCRIPTION

[0098] The present invention will be described in detail below in conjunction with specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly presented. It should be understood by those skilled in the art that these specific embodiments and examples are used to illustrate the present invention, rather than to limit the present invention.

[0099] Example 1: Primers used in the present invention

[0100] The primer sets used in the present invention are shown in Table 1.

[0101] Table 1

[0102]

[0103]

[0104]

[0105] Example 2: Method for detecting different targets based on high-throughput sequencing

[0106] The specific library construction process is as follows Figure 1 shown.

[0107] Sample preparation

[0108] Use a diluted Mycobacterium tuberculosis DNA standard as a template, or use a commercial extraction kit to extract nucleic acid from clinical samples or Mycobacterium tuberculosis cultures.

[0109] First round of multiplex amplification

[0110] Using the extracted gDNA as a template, multiplex PCR amplification was performed using QIAGEN Multiplex PCR Kit amplification reagent and a mixed primer set. The amplification system is shown in Table 2, and the reaction procedure is shown in Table 3.

[0111] Table 2

[0112] Components Volume (μL) Input DNA 10 Mix 25 Q-Solution 10 Primer set 5

[0113] Table 3

[0114]

[0115]

[0116] PCR product purification

[0117] The multiplex PCR products were purified at 0.8× using VAHTS DNA Clean Beads (N411) from Novozymes and eluted with 30 μL ddH2O.

[0118] Determination of amplification product concentration

[0119] The multiplex amplification products were quantified using the Qubit 4.0 instrument.

[0120] Library construction

[0121] Use Yisheng's one-step library preparation kit Hieff NGS @ OnePotⅡDNALibrary Prep Kit for Illumina @ For library construction, 5-50 ng DNA was calculated and added according to the concentration of multiple amplification test products. The amplification system for enzyme digestion and end repair is shown in Table 4, and the reaction procedure is shown in Table 5.

[0122] Table 4

[0123]

[0124] Table 5

[0125] temperature time 4℃ 1 min 30℃ 8 min 72℃ 5 min 4℃ Hold

[0126] The adapter-ligated amplification system is shown in Table 6, and the reaction procedure is shown in Table 7.

[0127] Table 6

[0128] Components Volume (μL) dA-tailed DNA 60 Ligation Enhancer 30 PE Adapter 5 Fast T4 DNA Ligase 5

[0129] Table 7

[0130] temperature time 20℃ 5 min 4℃ Hold

[0131] The amplification system for library amplification is shown in Table 8, and the reaction procedure is shown in Table 9.

[0132] Table 8

[0133]

[0134] Table 9

[0135]

[0136] 2.6 Library purification

[0137] The library amplification product was purified 1× using VAHTS DNA Clean Beads (N411) (0.9× magnetic beads) and eluted with 30 μL ddH2O.

[0138] 2.7 Library Quantification

[0139] Same as 2.4

[0140] 2.6 Library quality detection

[0141] Qseq400 was used to detect the fragment distribution of the library.

[0142] 2.7 Sequencing

[0143] The sequencing was performed using the TrueMed FASTASeq300 and the matching 75cycles reagent. The sequencing mode was SE75+8+8. The data volume was 5M per sample.

[0144] Example 3: Test results of the test samples of the composition of the present invention

[0145] Using DNA standards as templates, amplification was performed using the composition described in Example 1 and the average depth results of each amplicon were obtained by second-generation sequencing. The results are shown in Tables 10 and 11. It can be seen from the table that the composition of the present invention can amplify all targets in one tube and has good uniformity.

[0146] Table 10

[0147]

[0148] Table 11

[0149]

[0150]

[0151] After the average depth is normalized to 1000, the amplicon depth map is as follows Figure 2 As shown in the figure, it can be seen that each amplicon fragment can be detected, and the average depth of the amplicon is between 0.4-1.6 times. And the depth of the corresponding site of each gene can be detected. Taking the pncA gene as an example, the specific detection situation is as follows Figure 3 shown.

[0152] Example 4, Drug-resistant gene mutation detection test

[0153] Nucleic acid was extracted from pure culture of strains confirmed by WGS to contain drug-resistant gene mutation sites (the gene mutation information is shown in Table 12), and the nucleic acid of the resistant strain and the synthetic plasmid containing the drug-resistant gene mutation sites were diluted to 100 copies / μL as templates. The detection was performed according to the standard process. Finally, the four mutations in Table 12 were all accurately detected by the composition of the present invention, indicating that the composition of the present invention is capable of detecting a variety of tuberculosis drug-resistant gene mutations.

[0154] Table 12

[0155] drug Sample Type Gene mutation type Mutation detection results Rifampin Drug-resistant strain nucleic acid rpoB_p.Ser450Leu Checkout Isoniazid Drug-resistant strain nucleic acid katG_p.Ser315Thr Checkout Ethambutol Drug-resistant strain nucleic acid embB_p.Met306Ile Checkout Pyrazinamide Synthetic plasmid pncA_p.His57Asp Checkout

Claims

1. A composition for confirming drug resistance of Mycobacterium tuberculosis, comprising a primer set for detecting at least 4 of the following targets: A primer set for amplifying the ahpC gene as shown in SEQ ID NOs. 1 to 2; A primer set for amplifying the atpE gene as shown in SEQ ID NOs. 3 to 4; A primer set for amplifying the ddn gene as shown in SEQ ID NOs. 5 to 6; A primer set for amplifying the eis gene as shown in SEQ ID NOs. 7 to 8; A primer set for amplifying the embB gene as shown in SEQ ID NOs. 9 to 10; A primer set for amplifying the erm gene as shown in SEQ ID NOs. 11 to 12; A primer set for amplifying the ethA gene as shown in SEQ ID NOs. 13 to 16; A primer set for amplifying the fabG1 gene as shown in SEQ ID NOs. 17 to 18; A primer set for amplifying the fbiA gene as shown in SEQ ID NOs. 19 to 20; A primer set for amplifying the fbiB gene as shown in SEQ ID NOs. 21 to 22; A primer set for amplifying the fbiC gene as shown in SEQ ID NOs. 23 to 28; A primer set for amplifying the fgd1 gene as shown in SEQ ID NOs. 29 to 30; A primer set for amplifying the gidB gene as shown in SEQ ID NOs. 31 to 32; A primer set for amplifying the gyrA gene as shown in SEQ ID NOs. 33 to 34; A primer set for amplifying the gyrB gene as shown in SEQ ID NOs. 35 to 36; A primer set for amplifying the inhA gene as shown in SEQ ID NOs. 37 to 38; A primer set for amplifying the katG gene as shown in SEQ ID NOs. 39 to 42; A primer set for amplifying the pepQ gene as shown in SEQ ID NOs. 43 to 46; A primer set for amplifying the pncA gene as shown in SEQ ID NOs. 47 to 48; A primer set for amplifying the rplC gene as shown in SEQ ID NOs. 49 to 50; A primer set for amplifying the rpoB gene as shown in SEQ ID NOs. 51 to 52; A primer set for amplifying the rpsA gene as shown in SEQ ID NOs. 53 to 56; A primer set for amplifying the rpsL gene as shown in SEQ ID NOs. 57 to 58; A primer set for amplifying the rrL gene as shown in SEQ ID NOs. 59 to 60; A primer set for amplifying the rrs gene as shown in SEQ ID NOs. 61 to 62; A primer set for amplifying the rv0678 gene as shown in SEQ ID NOs. 63-64; A primer set for amplifying the Rv1979c gene as shown in SEQ ID NOs. 65 to 68; A primer set for amplifying the Rv2983 gene as shown in SEQ ID NOs. 69 to 70; A primer set for amplifying the tlyA gene as shown in SEQ ID NOs. 71 to 72; or The primer set for amplifying the ubiA gene is shown in SEQ ID NOs. 73-74.

2. The composition according to claim 1, characterized in that A primer set is included for detecting at least 15 of the above targets.

3. The composition according to claim 2, characterized in that A primer set for detecting at least 20 of the above targets is included.

4. The composition according to claim 3, characterized in that include: A primer set for amplifying the ahpC gene as shown in SEQ ID NOs. 1 to 2; A primer set for amplifying the atpE gene as shown in SEQ ID NOs. 3 to 4; A primer set for amplifying the ddn gene as shown in SEQ ID NOs. 5 to 6; A primer set for amplifying the eis gene as shown in SEQ ID NOs. 7 to 8; A primer set for amplifying the embB gene as shown in SEQ ID NOs. 9 to 10; A primer set for amplifying the erm gene as shown in SEQ ID NOs. 11 to 12; A primer set for amplifying the ethA gene as shown in SEQ ID NOs. 13 to 16; A primer set for amplifying the fabG1 gene as shown in SEQ ID NOs. 17 to 18; A primer set for amplifying the fbiA gene as shown in SEQ ID NOs. 19 to 20; A primer set for amplifying the fbiB gene as shown in SEQ ID NOs. 21 to 22; A primer set for amplifying the fbiC gene as shown in SEQ ID NOs. 23 to 28; A primer set for amplifying the fgd1 gene as shown in SEQ ID NOs. 29 to 30; A primer set for amplifying the gidB gene as shown in SEQ ID NOs. 31 to 32; A primer set for amplifying the gyrA gene as shown in SEQ ID NOs. 33 to 34; A primer set for amplifying the gyrB gene as shown in SEQ ID NOs. 35 to 36; A primer set for amplifying the inhA gene as shown in SEQ ID NOs. 37 to 38; A primer set for amplifying the katG gene as shown in SEQ ID NOs. 39 to 42; A primer set for amplifying the pepQ gene as shown in SEQ ID NOs. 43 to 46; A primer set for amplifying the pncA gene as shown in SEQ ID NOs. 47 to 48; A primer set for amplifying the rplC gene as shown in SEQ ID NOs. 49 to 50; A primer set for amplifying the rpoB gene as shown in SEQ ID NOs. 51 to 52; A primer set for amplifying the rpsA gene as shown in SEQ ID NOs. 53 to 56; A primer set for amplifying the rpsL gene as shown in SEQ ID NOs. 57 to 58; A primer set for amplifying the rrL gene as shown in SEQ ID NOs. 59 to 60; A primer set for amplifying the rrs gene as shown in SEQ ID NOs. 61 to 62; A primer set for amplifying the rv0678 gene as shown in SEQ ID NOs. 63-64; A primer set for amplifying the Rv1979c gene as shown in SEQ ID NOs. 65 to 68; A primer set for amplifying the Rv2983 gene as shown in SEQ ID NOs. 69 to 70; A primer set for amplifying the tlyA gene as shown in SEQ ID NOs. 71 to 72; and The primer set for amplifying the ubiA gene is shown in SEQ ID NOs. 73-74.

5. The composition according to claim 4, characterized in that The components of the composition are present in admixture.

6. Use of the composition according to any one of claims 1 to 5 for preparing a kit for confirming drug resistance of Mycobacterium tuberculosis.

7. A kit for confirming the drug resistance of Mycobacterium tuberculosis, comprising the composition according to any one of claims 1 to 5.

8. The kit according to claim 7, characterized in that The kit also includes at least one of the following: a nucleic acid extraction reagent, a nucleic acid amplification reagent, a library construction reagent, and a sequencing reagent.

9. Use of a composition for preparing a reagent for confirming drug resistance of Mycobacterium tuberculosis, the detection comprising: 1) Extracting or releasing nucleic acid from the sample to be tested; 2) amplifying using the composition according to any one of claims 1 to 5 to obtain an amplified product; 3) Processing the amplified products and building a library; and 4) Sequencing and analyzing the results.

10. The use according to claim 9, characterized in that The conditions for the amplification are:

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