A method, primer set and kit for capturing the whole genome of Mycobacterium tuberculosis

Abstract

This invention discloses a method, primer set, and kit for capturing the whole genome of Mycobacterium tuberculosis. The primer combination disclosed in this invention can effectively amplify the whole genome sequence of Mycobacterium tuberculosis with high coverage, which is beneficial for further research on the pathogenic mechanism, molecular epidemiology, and drug resistance mechanism of Mycobacterium tuberculosis.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a method for capturing the whole genome of Mycobacterium tuberculosis, a primer set, and a reagent kit. Background Technology

[0002] Mycobacterium tuberculosis (MTB) is the pathogen of tuberculosis. Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis, which seriously endangers human health and social development. Current tuberculosis prevention and control efforts face enormous challenges. Accurate diagnosis of Mycobacterium tuberculosis infection plays a crucial role in tuberculosis prevention and control. Direct whole-genome sequencing (WGS) of raw clinical samples can provide accurate sequences within 24-48 hours, accurately and promptly identifying bacterial gene mutation sites, discovering new mutation sites, and applying to epidemiology and MTB drug resistance mechanism research. Although the drug resistance mechanism of MTB is not fully understood, and there is no complete database for drug susceptibility testing and phenotypic prediction after genome sequencing, whole-genome sequencing can be integrated into routine diagnostic workflows to gradually replace drug susceptibility and phenotypic testing techniques in early diagnostic detection.

[0003] Whole genome sequencing (WGS), a commonly used genotyping technique, boasts high resolution. It can not only identify single nucleotide polymorphisms (SNPs) in the genome to determine the microevolution of mesenchymal tuberculosis (MTB) during transmission, but also obtain the complete sequence of the target genome, predicting resistance to most anti-tuberculosis drugs. NGS is widely used in MTB resistance detection due to its simplicity, speed, accuracy, and reliability, particularly in detecting multidrug-resistant tuberculosis patients, and is crucial for the effective treatment of drug-resistant Mycobacterium tuberculosis.

[0004] Whole-genome sequencing (WGGS) technology obtains the complete MTB genome sequence, and by utilizing known MTB resistance mechanisms, related resistance mutations can be detected at the whole-genome level. This expands molecular biology-based resistance detection beyond just one or a few first-line drugs. WGGS is frequently used in scientific research on drug resistance to discover new resistance sites and elucidate novel resistance mechanisms.

[0005] Therefore, obtaining the complete genome of MTB is of great significance. Summary of the Invention

[0006] To overcome the shortcomings of existing technologies, the present invention aims to provide a method, primer set, and kit for capturing the whole genome of Mycobacterium tuberculosis.

[0007] To achieve the above objectives, the present invention adopts the following technical solution:

[0008] The first aspect of this invention provides a primer combination for amplifying the whole genome of Mycobacterium tuberculosis.

[0009] Furthermore, the primer set includes 20 primers designed for the Mycobacterium tuberculosis cell genome, and the 20 primers have the sequences shown in TTCGTCGA, CGATACCG, CGATCGTC, CTTCGTCG, CGAAGTCG, CGAATCCG, CGAACTCG, CGATTCCG, CGATCAGC, GTTGACCG, GTTGTCGG, GTCGTCG, ATCGTCG, TCGTCGG, CGAACCG, GCGACG, CGTACCG, CGTAGCG, CGTTCGTA, and TATCGCG.

[0010] In this invention, primer combinations and primers are interchangeable. Primers refer to short nucleic acid molecules, and the primers disclosed in this invention are between 7 and 8 nucleotides in length. A hybrid can be formed between the primer and the target nucleic acid strand through nucleic acid hybridization and annealing with a complementary target nucleic acid molecule. The primer can be extended along the target nucleic acid molecule using polymerase. Therefore, primers can be used to amplify target nucleic acid molecules, wherein the primer sequence is specific to the target nucleic acid molecule.

[0011] A target nucleic acid molecule is a nucleic acid molecule intended for detection, quantification, qualitative analysis, or a combination thereof. The nucleic acid molecule does not necessarily have to be in purified form. Various other nucleic acid molecules can coexist with the target nucleic acid molecule. For example, the target nucleic acid molecule can be a specific nucleic acid molecule intended for amplification. If necessary, the purification or isolation of the target nucleic acid molecule can be performed using methods known to those skilled in the art, such as using commercially available purification kits.

[0012] In some embodiments, primers and / or probes corresponding to sequences having more than 70% homology with the primers and / or probe sequences described in this invention are also included within the scope of protection of this invention. That is, it can be understood that primers and / or probes obtained by modifying the primers and / or probes provided in this invention also fall within the scope of protection of this invention.

[0013] A second aspect of the present invention provides a reagent kit.

[0014] Furthermore, the kit includes the primer combination described in the first aspect of the present invention.

[0015] Furthermore, the kit also includes PCR amplification buffer, amplification enzyme, DTT, dNTP, pyrophosphatase, and nuclease-free purified water.

[0016] Furthermore, the amplification enzyme includes DNA polymerase and / or RNA polymerase.

[0017] Preferably, the amplification enzyme is a DNA polymerase.

[0018] Furthermore, the kit also includes instructions.

[0019] In this invention, suitable amounts of one or more primers are provided in one or more containers or immobilized on a matrix. The primers may be provided as a suspension in an aqueous solution or, for example, as a lyophilized or freeze-dried powder. The container providing the nucleic acid can be any conventional container capable of containing the provided form, such as a microcentrifuge tube, ampoule, or bottle. The kit may contain labeled or unlabeled probes for detecting Mycobacterium tuberculosis sequences.

[0020] In some applications, one or more primers (as described above) can be provided in pre-measured, single-use amounts in separate, typically disposable tubes or equivalent containers. Using such a setup, samples for testing the presence of gastrointestinal viruses can be added to separate tubes for direct amplification.

[0021] The amount of nucleic acid primers provided in the kit can be any suitable amount, depending on the target market for the product. For example, if the kit is intended for research or clinical applications, the amount of each nucleic acid primer provided can be sufficient to initiate several PCR amplification reactions. General guidelines for determining suitable amounts can be found in the literature of Innis et al., Sambrook et al., and Ausubel et al. The kit may contain more than two primers to facilitate PCR amplification of larger quantities of Mycobacterium tuberculosis nucleotide sequences.

[0022] In some implementations, the kit may contain reaction reagents necessary for performing PCR amplification, including DNA sample preparation reagents, enzymes for PCR, buffers, and Mg2+. 2+ and deoxyribonucleotides (dNTPs).

[0023] The enzymes used in PCR include DNA polymerase and / or RNA polymerase.

[0024] The DNA polymerases include, but are not limited to, Taq, Bst, Vent, Phi29, Pfu, Tru, Tth, Tl1, Tac, Tne, Tma, Tih, Tf1, Pwo, Kod, Sac, Sso, Poc, Pab, Mth, Pho, ES4 DNA polymerase, and the Klenow fragment.

[0025] dNTPs are nucleoside sources for PCR-based DNA amplification; dATP, dGTP, dCTP, and dTTP are essential. Additionally, for dNTPs, chemically modified substances designed for hot-start methods can be used, such as CleanAmp from TriLink BioTechnologies, Inc. TM dNTP.

[0026] In PCR-based DNA amplification, Mg 2+ It is necessary. As Mg 2+ Sources, including but not limited to MgCl2, MgSO4, etc.

[0027] In this invention, the kit also includes an instruction manual, which may include instructions on obtaining and processing samples.

[0028] In addition, the kit may contain bacterial genomic DNA as a positive control for PCR and sterile water as a negative control.

[0029] When implementing this invention, other necessary equipment can include pipettes, pipette tips, 1.5ml microtubes, and other instruments widely used in molecular biology experiments. As for devices, PCR machines, clean benches, tube centrifuges, and other instruments widely used in molecular biology experiments can be cited.

[0030] A third aspect of the present invention provides a method for obtaining the whole genome sequence of Mycobacterium tuberculosis.

[0031] Furthermore, the method uses the primer combination described in the first aspect of the present invention to amplify the whole genome sequence of Mycobacterium tuberculosis.

[0032] Furthermore, the method includes: performing PCR amplification on the sample using the primer combination described in the first aspect of the present invention, recovering and purifying the amplification product, library amplification, library purification, library quality control, and sequencing.

[0033] Furthermore, based on a total volume of 18 μL, the PCR amplification system includes: 2 μL Reaction Mix, 5 μL Primer Mix, 2 μL dNTP Mix, 2 μL DTT, 1 ng to 1 μg DNA sample, and the remainder is water.

[0034] Furthermore, after incubating the above PCR amplification system at 95°C for 5 min, 1 μL of DNA Polymerase and 1 μL of Pyrophosphatase were added.

[0035] Furthermore, after adding the reagents described above, the PCR amplification reaction program is 42℃ for 2 hours; 65℃ for 10 minutes; and stored at 4℃.

[0036] Furthermore, the samples include sputum, throat swabs, body fluids, secretions, puncture fluid, feces, etc.

[0037] In this invention, those skilled in the art will understand that, with appropriate adjustments to the reaction conditions and / or the amounts of each substance in the amplification reaction, the purpose of detecting Mycobacterium tuberculosis in the test sample from the subject can still be achieved. Therefore, the reaction conditions and / or the amounts of each substance in the amplification reaction described above are not intended to limit the scope of protection of this invention. With appropriate adjustments, as long as the purpose of detecting Mycobacterium tuberculosis in the test sample can be achieved, the adjusted reaction conditions and / or the amounts of each substance will also fall within the scope of protection of this invention.

[0038] In some implementations, the sample is derived from a clinical sample of a subject in need, including but not limited to: cells, tissues, body fluids, such as skin; mucous membranes; blood; blood derivatives or fractions, such as serum; extracted bile; tissue obtained by biopsy or surgery, including, for example, unfixed, frozen, formalin-fixed and / or paraffin-embedded tissue; tears; breast milk; skin flakes; surface cleaning solution; urine; sputum; cerebrospinal fluid; prostatic fluid; pus; bone marrow aspirate; middle ear effusion, bronchoalveolar lavage fluid, tracheal aspirate, sputum, nasopharyngeal aspirate, oropharyngeal aspirate, or saliva.

[0039] In some implementations, library amplification can be divided into two main categories: temperature-dependent amplification and temperature-dependent amplification. Temperature-dependent amplification mainly includes classic polymerase chain reaction (PCR) and ligase chain reaction (LCR), while temperature-dependent amplification includes strand displacement amplification (SDA), rolling circle amplification (RCA), loop-mediated amplification (LAMP), helicase-dependent isothermal DNA amplification (HDA), nucleic acid sequence-based amplification (NASBA), and transcription-based amplification systems (TAS). In this specific embodiment, PCR is used as an example for library construction.

[0040] In some embodiments, primers containing a tag sequence (index) may be used in the library amplification. By amplifying the ligation product using primers containing the tag sequence, data from different sample sources in sequencing can be distinguished, and the sample origin can be identified. The design method of the tag sequence and the design of primers containing the tag sequence (index) are both commonly known to those skilled in the art.

[0041] In some implementations, the sequencing can be performed using high-throughput sequencing platforms including Illumina NovaSeq, HiSeq X Ten, Illumina HiSeq, Illumina MiSeq, PacBio Sequel, 10×Genomics, and MGISEQ-2000.

[0042] In a specific embodiment of the present invention, the sequencing is performed using the Illumina MiSeq platform.

[0043] A fourth aspect of the present invention provides a library for detecting Mycobacterium tuberculosis.

[0044] Furthermore, the library includes the primer combination described in the first aspect of the present invention.

[0045] In some embodiments, a library refers to a collection of DNA or RNA samples containing a large number of gene sequences. These samples are collected, classified, cataloged, and labeled so that researchers can easily find and use them to study gene function or search for clones of specific genes. The process of establishing a library is similar to establishing a library, both aimed at efficiently managing and utilizing large amounts of information or material resources. In the detection of Mycobacterium tuberculosis, a library may contain multiple gene sequences associated with the bacterium, contributing to a deeper understanding of its biological characteristics, pathogenic mechanisms, and drug resistance.

[0046] The fifth aspect of this invention provides the application of the primer combination described in the first aspect of this invention in the preparation of products that amplify or detect Mycobacterium tuberculosis nucleic acid sequences.

[0047] The sixth aspect of this invention provides the application of the primer combination described in the first aspect of this invention or the kit described in the second aspect of this invention in amplifying the whole genome sequence of Mycobacterium tuberculosis or in Mycobacterium tuberculosis sequencing.

[0048] Compared with existing technologies, the present invention has the following advantages and benefits: the primer combination designed in the present invention has high amplification efficiency and uniform genome coverage. Detailed Implementation

[0049] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0050] In each of the following embodiments, the equipment and materials used are sourced from the following sources:

[0051] Bacterial DNA Extraction Kit: Tianlong Bacterial Genomic DNA Extraction Kit

[0052] Instrument: Thermo Qubit instrument (part number: Q33238)

[0053] Reagent: Thermo Qubit 1X dsDNAHS (High Sensitivity) Quantitative Reagent Kit (Catalog No.: Q33231)

[0054] illumina DNAPrep Library Preparation Kit (Catalog No.: TD502)

[0055] Example 1: Obtaining the complete genome of Mycobacterium tuberculosis

[0056] 1. Experimental materials

[0057] The reagent components used in the experiment, namely Reaction Mix, DNA Polymerase, and DTT, were derived from Thermo Fisher's EquiPhi29. TM DNA Polymerase (Catalog No.: A39390); dNTP mix from Thermo Fisher (Catalog No.: R0192); Pyrophosphatase from Thermo Fisher (Catalog No.: EF0221); Nucleasa-free water from Thermo Fisher (Catalog No.: 2305233).

[0058] The primer sequence information used in the experiment is shown in Table 1, and was synthesized by Sangon Biotech.

[0059] Table 1 Primer sequence information

[0060]

[0061]

[0062] 2. Nucleic acid extraction from samples:

[0063] Extract the DNA according to the requirements and steps in the bacterial DNA extraction kit. The recommended final DNA elution volume is 20-50 μL. The extracted tuberculosis DNA can be used directly for sequencing detection. If the extracted tuberculosis DNA is not used for direct detection, it can be stored at -80℃ for later use, but repeated freeze-thaw cycles should be avoided.

[0064] 3. Whole genome capture:

[0065] (1) Place each component of this kit on ice or thaw at 4°C in advance, mix by inverting, and then centrifuge briefly.

[0066] (2) Add the following reagents to a 0.2 mL thin-walled PCR tube containing no nucleic acid while on ice:

[0067] Table 2 Reagent Components

[0068]

[0069] (3) After gently mixing the above reaction solution, briefly centrifuge and incubate at 95°C for 5 min. After the incubation, place the product on ice immediately.

[0070] (4) Add the following reagents to the above mixture on ice:

[0071] Table 3 Reagents

[0072]

[0073] (5) After gently mixing the above reaction solution, briefly centrifuge and then proceed with the amplification according to the following procedure:

[0074] Table 4 Primer amplification procedure

[0075]

[0076] (6) Add 40 μL of ddH2O to the amplified product, mix gently, and centrifuge briefly before purification. If no further experiments are to be performed, the product can be stored at -20℃ for later use.

[0077] 4. Product recycling

[0078] It is recommended to use 1.8 times the volume of Beijing MicroFuture purification magnetic beads to purify the above amplification products. The purification magnetic beads need to be equilibrated at room temperature for 30 minutes and then shaken to ensure full activation; otherwise, the purification recovery rate will be affected. The purified product can be used for subsequent library construction. If not used directly for subsequent experiments, it can be stored at -20℃ for later use.

[0079] 5. Purification

[0080] After the amplification reaction is completed, varicella-zoster virus gene fragments of different sizes will be obtained from one PCR reaction tube. After the experiment is completed, the amplification products from 5 tubes are concentrated into a 1.5mL EP tube, purified, and quantified by qubit before proceeding directly to the subsequent library construction experiment.

[0081] (1) Add 250 μL AMPure XPbeads; *Note: This step is 1.0x-1.5x AMPure XPbeads.

[0082] (2) Shake at 1800 rpm for 2 min; incubate for 5 min;

[0083] (3) Let stand on the magnetic rack for 2 minutes or until it clarifies, then discard the supernatant;

[0084] (4) Add 500 μL of freshly prepared 80% ethanol, let stand on a magnetic rack for 30 seconds, and discard the supernatant;

[0085] (5) Repeat the above steps;

[0086] (6) Remove residual liquid using a 10μL pipette tip;

[0087] (7) Dry on a magnetic rack for 2 minutes;

[0088] (8) Remove from the magnetic rack; add 40 μL RSB;

[0089] (9) Shake at 1800 rpm for 2 min, then incubate at room temperature for 2 min;

[0090] (10) Let it stand on the magnetic rack until the liquid becomes clear;

[0091] (11) Transfer 38 μL of supernatant to a new plate.

[0092] 6. Quantitative

[0093] Operation procedure (refer to the document: QUICK REFERENCE-Qubit) TM Assays(MAN0017210vA.0):

[0094] a) Mix 1 μL of dye reagent with 199 μL of buffer solution by vortexing to obtain the working solution, and store in the dark until ready for use.

[0095] b) Take 1-20 μL of the library into the Qubit working solution, with a final volume of 200 μL.

[0096] c) Vortex for 2-3 seconds, then place at room temperature away from light for 2 minutes.

[0097] d) Use Qubit to perform measurements.

[0098] Example 2: Library Construction Operation Process

[0099] 1. DNA fragmentation

[0100] (1) Qubit quantification: Add 2-30 μL DNA (1-500 ng) to a PCR tube;

[0101] *Note: When using multiple samples, the concentration should ideally be greater than 50 or less than 50 ng to facilitate subsequent PCR amplification.

[0102] (2) Add pure water to the sample wells to bring the total volume to 30 μL;

[0103] (3) Vortex BLT10s, check whether the magnetic bead is completely suspended;

[0104] (4) Prepare the fragmented master reaction solution. For each reaction: BLT 11μL + TB 111μL, vortex the master reaction solution to mix well.

[0105] (5) Add 20 μL of the main reaction solution to the PCR sample well and vortex to mix (total 50 μL);

[0106] (6) Place in a PCR instrument and run the TAG Program (reaction system: 50 μL / well): heat the lid at 100℃; 55℃ for 15 min; store at 10℃.

[0107] 2. Purification

[0108] (1) Add 10 μL TSB to the fragmentation reaction solution, gently blow and mix 10 times, and then suspend the beads again;

[0109] *Note: TSB (check for precipitation) and TWB should be stored at 15-30℃ and brought to room temperature before use.

[0110] (2) Run the program in the PCR instrument: (set the hot lid to 100℃, the system to 60μL); 37℃, 15min; store at 10℃;

[0111] (3) Place the reaction tube in the magnetic rack and wait for the liquid to become clear (about 3 minutes). Then, remove and discard the supernatant.

[0112] (4) Remove the reaction tube from the magnetic rack, slowly add 100 μL of TWB, resuspend the magnetic beads, put the reaction tube back into the magnetic rack, wait for the liquid to become clear (about 3 min), and then remove and discard the supernatant.

[0113] (5) Repeat step (4) once;

[0114] (6) Remove the reaction tube from the magnetic rack, slowly add 100 μL of TWB, resuspend the magnetic beads, put the reaction tube back into the magnetic rack, and wait for the liquid to become clear (about 3 min); keep the reaction tube on the magnetic rack until step (2) of amplification, fragmentation and tagging of DNA is performed.

[0115] 3. Amplification of fragmented and tagged DNA

[0116] 3.1 Preparation of PCR master reaction solution (per sample): 22 μL EPM + 22 μL water, vortex to mix, and shake gently;

[0117] *Note: EPM: Thaw on ice. Invert to mix and then briefly centrifuge;

[0118] DNAAdapter: Thaw at room temperature. For test tubes, vortex and then briefly centrifuge. For plate packs, briefly centrifuge before use.

[0119] 3.2 Remove the supernatant from the magnetic rack and immediately add 40 μL of PCR master reaction solution to the sample wells and mix well;

[0120] 3.3 For IndexAdapter Tubes, add 5 μL of i7 adapter and 5 μL of i5 adapter respectively and mix thoroughly by pipetting;

[0121] 3.4 Run the PCR program: Set the hot lid to 100℃, the system to 50μL, and the PCR program is shown in Tables 5 and 6.

[0122] Table 5. Correspondence between DNA starting amount and PCR cycle number

[0123] Total DNA starting amount (ng) PCR cycle number (X) 1-9 12 10-24 8 25-49 6 50-99 5 100-500 5 Blood / saliva 5

[0124] Table 6 PCR Procedure

[0125]

[0126] 4. Library purification

[0127] 4.1 Centrifuge the reaction tube and place it in a magnetic rack, waiting for the liquid to clarify (about 5 minutes);

[0128] 4.2 Pipette and transfer 45 μL of supernatant into a new centrifuge tube;

[0129] 4.3 Fully oscillate and resuspend SPB;

[0130] *Note: SPB needs to be brought to room temperature and vortexed before use;

[0131] 4.4 For 100-500 ng of starting DNA, perform the following steps:

[0132] a. Add 40 μL H2O to the reaction tube containing the supernatant;

[0133] b. Add 45 μL of SPB to the reaction tube, mix thoroughly, and incubate at room temperature for 5 min;

[0134] c. Place the reaction tube on the magnetic rack and wait for the liquid to clarify (about 5 minutes);

[0135] d. During incubation, vortex the SPB thoroughly, then add 15 μL to a new centrifuge tube;

[0136] e. Transfer 125 μL of supernatant from the reaction tube on the magnetic rack to a new centrifuge tube (containing 15 μL of LPB) and mix thoroughly;

[0137] 4.5 For <100ng of starting DNA, perform the following steps: Add 81 μL of PB to the reaction tube containing the supernatant and mix thoroughly;

[0138] 4.6 Incubate at room temperature for 5 min, place the reaction tube on a magnetic rack, wait for the liquid to clarify (about 5 min), and then aspirate and discard the supernatant;

[0139] 4.7 Add 200 μL of freshly prepared 80% ethanol to the reaction tube, incubate for 30 seconds, discard the supernatant, and repeat this step once;

[0140] 4.8 Keep the reaction tube on the magnetic rack, remove and discard the residual ethanol in the reaction tube, and let it air dry for 5 minutes;

[0141] 4.9 Remove the reaction tube from the magnetic rack, add 32 μL LSB to resuspend the magnetic beads, and incubate at room temperature for 2 min;

[0142] 4.10 Place the reaction tube on a magnetic rack and wait for the liquid to clarify (about 2 minutes). Transfer 30 μL of supernatant to a new centrifuge tube.

[0143] 5. Document Quality Control

[0144] 5.1 Take 1 μL of the constructed library and run it on an Agilent Bioanalyzer 2100 (optional);

[0145] 5.2 Qubit quantification;

[0146] Molar concentration (nM) = Mass concentration (ng / μL) * 10^6 / (660 * average fragment length of the library)

[0147] Example 3: MiSeq hands-on operation

[0148] I. Diluted and denatured libraries

[0149] 1) Recommended concentrations for use are shown in Table 7.

[0150] Table 7 Recommended Injection Concentration

[0151] Reagents and types for instrument use Initial concentration of sample Concentration range for library testing MiSeqv3 reagent kit 4nM 6-20pM MiSeqv2 reagent kit 4nM 6-20pM MiSeqv2 reagent kit 2nM 6-10pM

[0152] 2) Experimental preparation:

[0153] 1NNaOH, PCR-grade water, HT1, pipettes and tips, vortex mixer, handheld centrifuge.

[0154] 3) Operating steps:

[0155] 1.20 μL of 1N NaOH + 80 μL of ultrapure water yields 0.2N NaOH.

[0156] 2. Vortex oscillate for 3-5 seconds, then momentarily centrifuge, and repeat once.

[0157] 3.5 μL of 4 nM or 2 nM library + 5 μL of 0.2N NaOH.

[0158] 4. Vortex oscillate for 3-5 seconds, then momentarily centrifuge, and repeat once.

[0159] 5. Denature at room temperature for 5-10 minutes.

[0160] 6. HT1990μL pre-cooled with ice.

[0161] 7. Vortex for 5-10 seconds, then momentarily centrifuge. Repeat once.

[0162] 8. Based on the library type and optimized concentration, perform a second dilution according to the following table:

[0163] Table 8. 4nM Initial Concentration Library

[0164] On-machine concentration 6pM 8pM 10pM 12pM 15pM 20pM 20pM Library 180μL 240μL 300μL 360μL 450μL 600μL Ice precooling HT1 420μL 360μL 300μL 240μL 450μL 0μL

[0165] Table 9. Library of 2 nM starting concentrations

[0166] On-machine concentration 6pM 8pM 10pM 20pM Library 360μL 480μL 600μL Ice precooling HT1 240μL 120μL 0μL

[0167] 9. Place the diluted and denatured library on ice or in a refrigerator at 2-8°C, ready for use.

[0168] II. Experimental Results

[0169] Sixteen experimental groups were designed based on the Mycobacterium tuberculosis cell genome sequence. Sequencing experiments were performed on all 16 experimental groups, and the bioinformatics analysis results of the sequencing results are shown in the table below.

[0170] Table 10 Sequencing Results

[0171]

[0172]

[0173] In the primer names used in this invention: M represents Mycobacterium tuberculosis; R represents random primer; T represents designed primer; -50: final primer concentration of 50 μM; for example: MR-50: random primer volume of 50 μM; MT10-50: 10 designed primers of 50 μM; MT15-100: 15 designed primers of 100 μM.

[0174] 37rv refers to the reference Mycobacterium tuberculosis sequence name in this experiment, which is h37rv, with a full length of 4474617 bp. For example, 2.43 indicates that the alignment rate of the experimental group MR-50 with the reference Mycobacterium tuberculosis genome h37rv reads is 2.43%, meaning that the alignment rate of the gene sequence reads obtained using this set of primers with the reference Mycobacterium tuberculosis genome h37rv reads is 2.43%.

[0175] human: Refer to the human genome. For example, 20.41 indicates that the alignment rate of the human genome with the experimental group MR-50 is 20.41%, meaning that the alignment rate of the gene sequence reads obtained using this set of primers with the reference human genome reads is 20.41%.

[0176] Coverage: Based on the full-length (4474617 bp) Mycobacterium tuberculosis sequence name h37rv, this indicates the number of bases covered by the sequence obtained from the experimental group. For example, 3514115 indicates that the coverage of MR-50 in the experimental group is 3514115 bp, meaning that the gene sequence obtained using this set of primers covers 3514115 bp of the Mycobacterium tuberculosis sequence name h37rv.

[0177] Summary: According to the data in the table above, primer group 8, MT20-50, yielded the best experimental results. This primer group, with 20 primers and an input volume of 50 μM, achieved a tuberculosis genome alignment rate of 10.36%, a human genome alignment rate of 11.00%, and a coverage of 4,165,768 bp.

[0178] The above description of the embodiments is only for understanding the method and core ideas of the present invention. It should be noted that those skilled in the art can make various improvements and modifications to the present invention without departing from the principles of the invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. A primer combination for amplifying the whole genome of Mycobacterium tuberculosis, characterized in that, The primer combination consists of 20 primers designed for the Mycobacterium tuberculosis cell genome, and the sequences of the 20 primers are as follows: TTCGTCGA, CGATACCG, CGATCGTC, CTTCGTCG, CGAAGTCG, CGAATCCG, CGAACTCG, CGATTCCG, CGATCAGC, GTTGACCG, GTTGTCGG, GTCGTCG, ATCGTCG, TCGTCGG, CGAACCG, GCGACG, CGTACCG, CGTAGCG, CGTTCGTA, TATCGCG.

2. A reagent kit, characterized in that, The kit comprises the primer combination as described in claim 1.

3. The reagent kit according to claim 2, characterized in that, The kit also includes PCR amplification buffer, amplification enzyme, DTT, dNTP, pyrophosphatase, and nuclease-free purified water.

4. The reagent kit according to claim 3, characterized in that, The amplification enzymes include DNA polymerase and / or RNA polymerase.

5. The reagent kit according to claim 4, characterized in that, The amplification enzyme is a DNA polymerase.

6. The reagent kit according to claim 2, characterized in that, The kit also includes instructions.

7. A method for obtaining the complete genome sequence of Mycobacterium tuberculosis in vitro for non-therapeutic and non-diagnostic purposes, characterized in that, The method uses the primer combination described in claim 1 to amplify the whole genome sequence of Mycobacterium tuberculosis.

8. The method according to claim 7, characterized in that, The method includes: performing PCR amplification on the sample using the primer combination described in claim 1, recovering and purifying the amplification product, library amplification, library purification, library quality control, and sequencing.

9. The method according to claim 8, characterized in that, The PCR amplification system, with a total volume of 18 μL, includes: 2 μL Reaction Mix, 5 μL Primer Mix, 2 μL dNTP Mix, 2 μL DTT, 1 ng to 1 μg DNA sample, and the remainder is water.

10. The method according to claim 9, characterized in that, After incubating the PCR amplification system at 95°C for 5 minutes, add 1 μL of DNA Polymerase and 1 μL of Pyrophosphatase.

11. The method according to claim 9 or 10, characterized in that, The PCR amplification reaction program was 42℃ for 2 hours; 65℃ for 10 minutes; and stored at 4℃.

12. The use of the primer combination of claim 1 in the preparation of products for amplifying or detecting Mycobacterium tuberculosis nucleic acid sequences.

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