Construction of Mycobacterium tuberculosis resistance detection system for first-line treatment drugs based on multiple pcr-ms mini-sequence technology
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摘要:
目的 构建一种基于多重PCR-质谱微测序技术的结核分枝杆菌耐药基因多位点检测方法和检测模块,实现结核分枝杆菌对一线抗结核药物的高通量快速检测,为结核病诊疗提供一种新型技术支撑。 方法 通过对5个SNP位点靶基因进行多重PCR扩增、单碱质量探针延伸及分子量测定,同时完成16个突变位点、26种突变型的检测,实现结核分枝杆菌对利福平、异烟肼、吡嗪酰胺和乙胺丁醇等一线治疗药物抗性的通量检测。 采用40例结核分枝杆菌样本、50例呼吸道感染患者咽拭子样本进行检测体系准确性及特异性验证。 结果 本研究构建了基于5重PCR-质谱联用的结核分枝杆菌对一线治疗药物的泛耐药检测方法,并开发了质谱配套检测系统。 采用该系统,结核分枝杆菌对利福平、异烟肼、吡嗪酰胺、乙胺丁醇抗性检测的准确率和特异性均为100%,且具有7 h内完成96个样本的检测通量。 结论 本研究构建的方法具有操作简便、低成本、高通量的特点,耐药位点检测全面且准确,同时具有扩展性,可根据需要增添新的突变位点,在结核分枝杆菌耐药性检测分析中具有良好的应用前景。 -
关键词:
- 结核分枝杆菌 /
- 一线治疗药物 /
- 耐药性 /
- 多重PCR-质谱微测序技术
Abstract:Objective To establish a multi-locus detection method and detection module for Mycobacterium tuberculosis drug resistance gene based on multiplex PCR-mass spectrometry mini-sequencing technology and to realize the high-throughput rapid detection of first-line anti-tuberculosis drugs by Mycobacterium tuberculosis, which provide a new technical support for tuberculosis diagnosis and treatment. Methods Through multiplex PCR amplification, single base mass probe extension and molecular weight determination of 5 SNP locus target genes, and the detection of 16 mutation sites and 26 mutant types were completed at the same time, the flux detection of the resistance of Mycobacterium tuberculosis to first-line therapeutic drugs such as rifampicin, isoniazid, pyrazinamide and ethambutol was realized. The accuracy and specificity of the detection system were verified by using 40 samples of Mycobacterium tuberculosis and throat swab samples from 50 patients with respiratory infection. Results In this study, a pan-drug resistance detection method for first-line therapeutic drugs of Mycobacterium tuberculosis based on 5-plex PCR-mass spectrometry was constructed, and a mass spectrometry supporting detection system was developed. With this system, the accuracy and specificity of Mycobacterium tuberculosis resistance detection of rifampicin, isoniazid, pyrazinamide, and ethambutol were all 100%, and it had a detection throughput of 96 samples in 7 hours. Conclusion The method constructed has the characteristics of simple operation, low cost and high throughput, comprehensive and accurate detection of drug resistance sites, and scalability, and can add new mutation sites as needed, which has good application prospects in the detection and analysis of drug resistance of Mycobacterium tuberculosis. -
表 1 结核分枝杆菌主要耐药基因突变位点
Table 1. Drug resistance gene mutation sites of Mycobacterium tuberculosis
抗菌药物 耐药基因 位置 突变类型 利福平 rpoB Q513P CAA→CCA D516Y GAC→TAC D516G/V GAC→GG/TC H526D/Y/N CAC→G/T/AAC H526L/R CAC→CTC H531L/W TCG→TTG 异烟肼 katG S315T/N AGC→AC/AC inhA −15 C→T 乙胺丁醇 embB M306V ATG→GTG M306I ATG→ATC/A/T Y319S/C TAT→TC/GT G406D GGC→GAC G406S GGC→AGC 吡嗪酰胺 pncA Q10P CAG→CCG H57D CAC→GAC T76P ACT→CCT 表 2 结核分枝杆菌耐药基因扩增引物序列
Table 2. Sequence of primers for drug resistance gene amplification of Mycobacterium tuberculosis
抗菌药物 基因 引物编号 引物序列(5'~3') 利福平 rpoB F1-rpoB acgttggatgAACCAGATCCGGGTCGGCAT R1-rpoB acgttggatgTAACCACGCCGTCGACCACCTT 异烟肼 katG F2-katG acgttggatgCTGGAGCAGATGGGCTTG R2-katG acgttggatgAGGTCAGTGGCCAGCATC fabG-inhA F3- fabG1 acgttggatgCCTCGCTGCCCAGAAAGGGA R3- fabG1 acgttggatgGTAACCAGGACTGAACGG 乙胺丁醇 embB F4-embB acgttggatgCGACGCCGTGGTGATATT R4-embB acgttggatgACCGCTCGATCAGCACAT 吡嗪酰胺 pncA F6-pncA acgttggatgGCGTCATGGACCCTATATC R6-pncA acgttggatgCTCGTCGACTCCTTCGAAG 表 3 结核分枝杆菌MPE探针序列及其碱基延伸
Table 3. MPE probe sequence and its base extension of Mycobacterium tuberculosis
耐药
基因突变位点 突变型 探针延伸序列(5'~3') 分子量(Da) 延伸1 分子量(Da) 延伸2 分子量(Da) 延伸3 分子量(Da) 延伸4 分子量(Da) rpoB Q513PL A/C/T TTCGGCACCAGCCAGCTGAGCC 6681.4 A 6978.4 C 6954.4 T 7023.4 D516Y(F) G/T GCACCAGCCAGCTGAGCCAATTCATG 7925.2 G 8238.2 T 8267.2 D516GV(R) A/G/T CGACAGCGGGTTGTTCTGG 5875.8 A 6217.8 G 6148.8 T 6172.8 H526DYN(F) C/G/T/A ACCCGCTGTCGGGGTTGACC 6110.0 C 6383.0 G 6423.0 T 6452.0 A 6407.0 H526LR(R) A/T/G GCCGACAGTCGGCGCTTG 5516.6 A 5858.6 T 5813.6 G 5789.6 H531LW C/T/G GACCCACAAGCGCCGACTGT 6072.0 C 6345.0 T 6414.0 G 6385.0 katG S315TN G/C/A TGTTCGTCCATACGACCTCGATGC 7279.8 G 7552.8 C 7592.8 A 7621.8 inhA −15 C/T GCATGGGTATGGGCCACTGACA 6800.4 C 7073.4 T 7142.4 embB M306V(F) A/G GTCGGACGACGGCTACATCCTGGGC 7684.0 A 7981.0 G 7997.0 M306I(R) G/C/A/T GGACGACGGCTACATCCTGGGCAT 7378.8 G 7691.8 C 7651.8 A 7675.8 T 7720.8 Y319SC A/C/G CCACGCCGGCTACATGTCCAACT 6929.6 A 7226.6 C 7202.6 G 7242.6 G406D(R) G/A CCAGCGAGCCGAGCGCGATGATG 7099.6 G 7372.6 A 7441.6 G406S(F) G/A CAACGGCCTGCGGCCGGAG 5839.8 G 6152.8 A 6136.8 pncA Q10P A/C GCGAGCCACCCTCGCAGAAGTCGTTC 7917.2 A 8259.2 C 8230.2 H57D C/G GGAATAGTCCGGTGTGCCGGAGAAGT 8116.2 C 8429.2 G 8389.2 T76P A/C TGGGATGGAAGTCCGCGCCGGGAG 7499.8 A 7841.8 C 7812.8 注: 表 4 研究所用结核分枝杆菌核酸样本验证结果
Table 4. Verification results of nucleic acid samples from Mycobacterium tuberculosis used in this research
序号 突变位点MPE延伸 利福平(RIF) 异烟肼(INH) 乙胺丁醇(EMB) 吡嗪酰胺(PZA) rpoBH526LR rpoBH526DYN rpoBQ513PL rpoBD516GV(R) rpoBH531LW rpoBD516Y(F) katGS315TN fabG-inhA embBY319SC embBG406D(R) embBM306I(R) embBM306V(F) embBG406S pncAT76P pncAH57D pncAQ10P MTB-01 A C A A C G G C A G G A G A C A MTB-02 A C A A C G Aa C A G G A G A C A MTB-03 A C A A C G Aa C A G G A G A C A MTB-04 A C A A C G Aa C A G G A G A C A MTB-05 A C Ta A C G G C A G G A G A C A MTB-06 A Ga A A C G G Ta A G G A G A C A MTB-07 A C A A C Ta Ca C A G G A G A C A MTB-08 A C A A C G G C A G G A G A C A MTB-09 A Ta A A C G Ca C A G G A G A C A MTB-10 A C A A C Ta Ca C A G G A G A C A MTB-11 A C A A C G G C A G G A G A C A MTB-12 A C A A C G G Ta A G G A G A C A MTB-13 A C A A Ta G Ca C A G G A Aa A C A MTB-14 A C A A C G Ca C A G G Ga G A C A MTB-15 A C A A C G Ca C A G G Ga G A C A MTB-16 A C A A Ta G G C Ga G G A G A C A MTB-17 A C A A Ta G G Ta A G G A G A C A MTB-18 A C A A Ta G Ca C A G Ca A G A C A MTB-19 A C A A Ta G G Ta A Aa G A G A C A MTB-20 A C A A Ta G Ca C A G Ca A G A C A MTB-21 A C A Ta C G G Ta A Aa G A G A C A MTB-22 A C A A C G G Ta A G G A G A C A MTB-23 A Ta A A C G Ca C A G G A G A C A MTB-24 A C A A Ga G Aa C A G G A G A C A MTB-25 A Aa A A C G G C A G G A G A C A MTB-26 A C A A C G G C A G G A G A C A MTB-27 A Ta A A C G G C A G G A G A C A MTB-28 A Ga A A C G Ca C A G G A G A C A MTB-29 Ta C A A C G Ca C A G G A G A C A MTB-30 A C A A Ta G G Ta A G G A G A C A MTB-31 A C A A Ta G G C Ga G G Ga G A C A MTB-32 A C A A Ta G G Ta A G G Ga G A C A MTB-33 Ta C A A C G Ca C A G G Ga G A C A MTB-34 A C A A Ta G Ca C A G Ca A G A C A MTB-35 A C A A Ta G Ca Ta A G G Ga G A C A MTB-36 Ta C A A C G Ca C A G G Ga G A C A MTB-37 A Ta A A C G Ca C A G G Ga G A C A MTB-38 A C A A Ta G G C A G Ca A G A C A MTB-39 A C A A Ta G Ca C A G Ca A G A C A MTB-40 Ga C A A C G Ca C A G G Ga G A C A 注:a. 表示突变型,其余为野生型 表 5 研究所用结核分枝杆菌核酸样本全基因组测序结果
Table 5. Whole genome sequencing results of nucleic acid samples from Mycobacterium tuberculosis used in this research
序号 突变位点全基因组测序结果 利福平 异烟肼 乙胺丁醇 吡嗪酰胺 rpoB katG embB pncA 526 513 516 531 315 −15 319 406 306 76 57 10 MTB-01 CAC CAA GAC TCG AGC C TAT GGC ATG ACT CAC CAG MTB-02 CAC CAA GAC TCG AACa C TAT GGC ATG ACT CAC CAG MTB-03 CAC CAA GAC TCG AACa C TAT GGC ATG ACT CAC CAG MTB-04 CAC CAA GAC TCG AACa C TAT GGC ATG ACT CAC CAG MTB-05 CAC CTAa GAC TCG AGC C TAT GGC ATG ACT CAC CAG MTB-06 GACa CAA GAC TCG AGC Ta TAT GGC ATG ACT CAC CAG MTB-07 CAC CAA TACa TCG ACCa C TAT GGC ATG ACT CAC CAG MTB-08 CAC CAA GAC TCG AGC C TAT GGC ATG ACT CAC CAG MTB-09 TACa CAA GAC TCG ACCa C TAT GGC ATG ACT CAC CAG MTB-10 CAC CAA TACa TCG ACCa C TAT GGC ATG ACT CAC CAG MTB-11 CAC CAA GAC TCG AGC C TAT GGC ATG ACT CAC CAG MTB-12 CAC CAA GAC TCG AGC Ta TAT GGC ATG ACT CAC CAG MTB-13 CAC CAA GAC TTGa ACCa C TAT AGCa ATG ACT CAC CAG MTB-14 CAC CAA GAC TCG ACCa C TAT GGC GTGa ACT CAC CAG MTB-15 CAC CAA GAC TCG ACCa C TAT GGC GTGa ACT CAC CAG MTB-16 CAC CAA GAC TTGa AGC C TGTa GGC ATG ACT CAC CAG MTB-17 CAC CAA GAC TTGa AGC Ta TAT GGC ATG ACT CAC CAG MTB-18 CAC CAA GAC TTGa ACCa C TAT GGC ATCa ACT CAC CAG MTB-19 CAC CAA GAC TTGa AGC Ta TAT GACa ATG ACT CAC CAG MTB-20 CAC CAA GAC TTGa ACCa C TAT GGC ATCa ACT CAC CAG MTB-21 CAC CAA GTCa TCG AGC Ta TAT GACa ATG ACT CAC CAG MTB-22 CAC CAA GAC TCG AGC Ta TAT GGC ATG ACT CAC CAG MTB-23 TACa CAA GAC TCG ACCa C TAT GGC ATG ACT CAC CAG MTB-24 CAC CAA GAC TGGa AACa C TAT GGC ATG ACT CAC CAG MTB-25 AACa CAA GAC TCG AGC C TAT GGC ATG ACT CAC CAG MTB-26 CAC CAA GAC TCG AGC C TAT GGC ATG ACT CAC CAG MTB-27 TACa CAA GAC TCG AGC C TAT GGC ATG ACT CAC CAG MTB-28 GACa CAA GAC TCG ACCa C TAT GGC ATG ACT CAC CAG MTB-29 CTCa CAA GAC TCG ACCa C TAT GGC ATG ACT CAC CAG MTB-30 CAC CAA GAC TTGa AGC Ta TAT GGC ATG ACT CAC CAG MTB-31 CAC CAA GAC TTGa AGC C TGTa GGC ATG ACT CAC CAG MTB-32 CAC CAA GAC TTGa AGC Ta TAT GGC GTGa ACT CAC CAG MTB-33 CTCa CAA GAC TCG ACCa C TAT GGC GTGa ACT CAC CAG MTB-34 CAC CAA GAC TTGa ACCa C TAT GGC ATCa ACT CAC CAG MTB-35 CAC CAA GAC TTGa ACCa Ta TAT GGC GTGa ACT CAC CAG MTB-36 CTCa CAA GAC TCG ACCa C TAT GGC GTGa ACT CAC CAG MTB-37 TACa CAA GAC TCG ACCa C TAT GGC GTGa ACT CAC CAG MTB-38 CAC CAA GAC TTGa AGC C TAT GGC ATCa ACT CAC CAG MTB-39 CAC CAA GAC TTGa ACCa C TAT GGC ATCa ACT CAC CAG MTB-40 CGCa CAA GAC TCG ACCa C TAT GGC GTGa ACT CAC CAG 注:a. 表示突变型,其余为野生型 -
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