基于多重PCR-质谱微测序技术的结核分枝杆菌对一线治疗药物耐药性检测系统构建

徐晓娜; 赵欣; 欧喜超; 宋衍燕; 赵雁林; 肖迪

doi:10.3784/jbjc.202303160107

基于多重PCR-质谱微测序技术的结核分枝杆菌对一线治疗药物耐药性检测系统构建

doi: 10.3784/jbjc.202303160107

徐晓娜^{1, 2, 3,},
赵欣^2,,
欧喜超⁴,
宋衍燕³,
赵雁林^4, ,,
肖迪^2, ,

1.
内蒙古科技大学包头医学院, 内蒙古包头 014040
2.
中国疾病预防控制中心传染病预防控制所, 传染病预防控制国家重点实验室, 北京 102206
3.
北京市朝阳区疾病预防控制中心, 北京 100021
4.
中国疾病预防控制中心结核病预防控制中心, 国家结核病参比实验室, 北京 102206

详细信息

作者简介:
徐晓娜，女，内蒙古自治区乌兰察布市人，硕士研究生在读，主要从事病原菌耐药性新型检测方法构建，Email：xuxiaona97@163.com

赵欣，女，山西省晋中市人，硕士，研究实习员，主要从事核酸质谱分析工作，Email：zhaoxin@icdc.cn

通讯作者:
赵雁林，Email：zhaoyanlin@icdc.cn

肖迪，Tel：010–58900704，Email: xiaodi@icdc.cn

中图分类号: R211
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出版历程
- 收稿日期: 2023-03-16
- 录用日期: 2023-05-04
- 网络出版日期: 2023-05-05

Construction of Mycobacterium tuberculosis resistance detection system for first-line treatment drugs based on multiple pcr-ms mini-sequence technology

Xu Xiaona^{1, 2, 3
,},
Zhao Xin^2
,,
Ou Xichao⁴,
Song Yanyan³,
Zhao Yanlin^{4
, ,},
Xiao Di^{2
, ,}

1.
Baotou Medical college, Baotou 014040, China
2.
National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Prevention, State Key Laboratory of Infectious Disease Prevention and Control. Beijing 102206, China
3.
Chaoyang District Center for Disease Control and Prevention, Beijing. Beijing 100021, China
4.
National Tuberculosis Reference Laboratory National Center for Tuberculosis Control and Prevention China CDC, Beijing 102206, China

More Information

Corresponding author: Zhao Yanlin, Email: zhaoyanlin@icdc.cn; Xiao Di, Email: xiaodi@icdc.cn

摘要

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。
- 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.
- Mycobacterium tuberculosis /
- Drug resistance /
- First-line drugs /
- Multiplex PCR mass spectrometry mini-sequencing technology

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图 1 结核分枝杆菌突变位点的原始质量探针延伸质谱图

注：A、B. MPE探针原始峰值；C～L. MPE探针突变位点延伸结果；M、N. 空白对照

Figure 1. Original mass probe extension mass spectrometry of the mutant site of Mycobacterium tuberculosis

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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
异烟肼	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

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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
利福平	rpoB	R1-rpoB	acgttggatgTAACCACGCCGTCGACCACCTT
异烟肼	katG	F2-katG	acgttggatgCTGGAGCAGATGGGCTTG
	katG	R2-katG	acgttggatgAGGTCAGTGGCCAGCATC
	fabG-inhA	F3- fabG1	acgttggatgCCTCGCTGCCCAGAAAGGGA
	fabG-inhA	R3- fabG1	acgttggatgGTAACCAGGACTGAACGG
乙胺丁醇	embB	F4-embB	acgttggatgCGACGCCGTGGTGATATT
乙胺丁醇	embB	R4-embB	acgttggatgACCGCTCGATCAGCACAT
吡嗪酰胺	pncA	F6-pncA	acgttggatgGCGTCATGGACCCTATATC
吡嗪酰胺	pncA	R6-pncA	acgttggatgCTCGTCGACTCCTTCGAAG

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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：

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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	A^a	C	A	G	G	A	G	A	C	A
MTB-03	A	C	A	A	C	G	A^a	C	A	G	G	A	G	A	C	A
MTB-04	A	C	A	A	C	G	A^a	C	A	G	G	A	G	A	C	A
MTB-05	A	C	T^a	A	C	G	G	C	A	G	G	A	G	A	C	A
MTB-06	A	G^a	A	A	C	G	G	T^a	A	G	G	A	G	A	C	A
MTB-07	A	C	A	A	C	T^a	C^a	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	T^a	A	A	C	G	C^a	C	A	G	G	A	G	A	C	A
MTB-10	A	C	A	A	C	T^a	C^a	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	T^a	A	G	G	A	G	A	C	A
MTB-13	A	C	A	A	T^a	G	C^a	C	A	G	G	A	A^a	A	C	A
MTB-14	A	C	A	A	C	G	C^a	C	A	G	G	G^a	G	A	C	A
MTB-15	A	C	A	A	C	G	C^a	C	A	G	G	G^a	G	A	C	A
MTB-16	A	C	A	A	T^a	G	G	C	G^a	G	G	A	G	A	C	A
MTB-17	A	C	A	A	T^a	G	G	T^a	A	G	G	A	G	A	C	A
MTB-18	A	C	A	A	T^a	G	C^a	C	A	G	C^a	A	G	A	C	A
MTB-19	A	C	A	A	T^a	G	G	T^a	A	A^a	G	A	G	A	C	A
MTB-20	A	C	A	A	T^a	G	C^a	C	A	G	C^a	A	G	A	C	A
MTB-21	A	C	A	T^a	C	G	G	T^a	A	A^a	G	A	G	A	C	A
MTB-22	A	C	A	A	C	G	G	T^a	A	G	G	A	G	A	C	A
MTB-23	A	T^a	A	A	C	G	C^a	C	A	G	G	A	G	A	C	A
MTB-24	A	C	A	A	G^a	G	A^a	C	A	G	G	A	G	A	C	A
MTB-25	A	A^a	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	T^a	A	A	C	G	G	C	A	G	G	A	G	A	C	A
MTB-28	A	G^a	A	A	C	G	C^a	C	A	G	G	A	G	A	C	A
MTB-29	T^a	C	A	A	C	G	C^a	C	A	G	G	A	G	A	C	A
MTB-30	A	C	A	A	T^a	G	G	T^a	A	G	G	A	G	A	C	A
MTB-31	A	C	A	A	T^a	G	G	C	G^a	G	G	G^a	G	A	C	A
MTB-32	A	C	A	A	T^a	G	G	T^a	A	G	G	G^a	G	A	C	A
MTB-33	T^a	C	A	A	C	G	C^a	C	A	G	G	G^a	G	A	C	A
MTB-34	A	C	A	A	T^a	G	C^a	C	A	G	C^a	A	G	A	C	A
MTB-35	A	C	A	A	T^a	G	C^a	T^a	A	G	G	G^a	G	A	C	A
MTB-36	T^a	C	A	A	C	G	C^a	C	A	G	G	G^a	G	A	C	A
MTB-37	A	T^a	A	A	C	G	C^a	C	A	G	G	G^a	G	A	C	A
MTB-38	A	C	A	A	T^a	G	G	C	A	G	C^a	A	G	A	C	A
MTB-39	A	C	A	A	T^a	G	C^a	C	A	G	C^a	A	G	A	C	A
MTB-40	G^a	C	A	A	C	G	C^a	C	A	G	G	G^a	G	A	C	A
注：a. 表示突变型，其余为野生型

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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	AAC^a	C	TAT	GGC	ATG	ACT	CAC	CAG
MTB-03	CAC	CAA	GAC	TCG	AAC^a	C	TAT	GGC	ATG	ACT	CAC	CAG
MTB-04	CAC	CAA	GAC	TCG	AAC^a	C	TAT	GGC	ATG	ACT	CAC	CAG
MTB-05	CAC	CTA^a	GAC	TCG	AGC	C	TAT	GGC	ATG	ACT	CAC	CAG
MTB-06	GAC^a	CAA	GAC	TCG	AGC	T^a	TAT	GGC	ATG	ACT	CAC	CAG
MTB-07	CAC	CAA	TAC^a	TCG	ACC^a	C	TAT	GGC	ATG	ACT	CAC	CAG
MTB-08	CAC	CAA	GAC	TCG	AGC	C	TAT	GGC	ATG	ACT	CAC	CAG
MTB-09	TAC^a	CAA	GAC	TCG	ACC^a	C	TAT	GGC	ATG	ACT	CAC	CAG
MTB-10	CAC	CAA	TAC^a	TCG	ACC^a	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	T^a	TAT	GGC	ATG	ACT	CAC	CAG
MTB-13	CAC	CAA	GAC	TTG^a	ACC^a	C	TAT	AGC^a	ATG	ACT	CAC	CAG
MTB-14	CAC	CAA	GAC	TCG	ACC^a	C	TAT	GGC	GTG^a	ACT	CAC	CAG
MTB-15	CAC	CAA	GAC	TCG	ACC^a	C	TAT	GGC	GTG^a	ACT	CAC	CAG
MTB-16	CAC	CAA	GAC	TTG^a	AGC	C	TGT^a	GGC	ATG	ACT	CAC	CAG
MTB-17	CAC	CAA	GAC	TTG^a	AGC	T^a	TAT	GGC	ATG	ACT	CAC	CAG
MTB-18	CAC	CAA	GAC	TTG^a	ACC^a	C	TAT	GGC	ATC^a	ACT	CAC	CAG
MTB-19	CAC	CAA	GAC	TTG^a	AGC	T^a	TAT	GAC^a	ATG	ACT	CAC	CAG
MTB-20	CAC	CAA	GAC	TTG^a	ACC^a	C	TAT	GGC	ATC^a	ACT	CAC	CAG
MTB-21	CAC	CAA	GTC^a	TCG	AGC	T^a	TAT	GAC^a	ATG	ACT	CAC	CAG
MTB-22	CAC	CAA	GAC	TCG	AGC	T^a	TAT	GGC	ATG	ACT	CAC	CAG
MTB-23	TAC^a	CAA	GAC	TCG	ACC^a	C	TAT	GGC	ATG	ACT	CAC	CAG
MTB-24	CAC	CAA	GAC	TGG^a	AAC^a	C	TAT	GGC	ATG	ACT	CAC	CAG
MTB-25	AAC^a	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	TAC^a	CAA	GAC	TCG	AGC	C	TAT	GGC	ATG	ACT	CAC	CAG
MTB-28	GAC^a	CAA	GAC	TCG	ACC^a	C	TAT	GGC	ATG	ACT	CAC	CAG
MTB-29	CTC^a	CAA	GAC	TCG	ACC^a	C	TAT	GGC	ATG	ACT	CAC	CAG
MTB-30	CAC	CAA	GAC	TTG^a	AGC	T^a	TAT	GGC	ATG	ACT	CAC	CAG
MTB-31	CAC	CAA	GAC	TTG^a	AGC	C	TGT^a	GGC	ATG	ACT	CAC	CAG
MTB-32	CAC	CAA	GAC	TTG^a	AGC	T^a	TAT	GGC	GTG^a	ACT	CAC	CAG
MTB-33	CTC^a	CAA	GAC	TCG	ACC^a	C	TAT	GGC	GTG^a	ACT	CAC	CAG
MTB-34	CAC	CAA	GAC	TTG^a	ACC^a	C	TAT	GGC	ATC^a	ACT	CAC	CAG
MTB-35	CAC	CAA	GAC	TTG^a	ACC^a	T^a	TAT	GGC	GTG^a	ACT	CAC	CAG
MTB-36	CTC^a	CAA	GAC	TCG	ACC^a	C	TAT	GGC	GTG^a	ACT	CAC	CAG
MTB-37	TAC^a	CAA	GAC	TCG	ACC^a	C	TAT	GGC	GTG^a	ACT	CAC	CAG
MTB-38	CAC	CAA	GAC	TTG^a	AGC	C	TAT	GGC	ATC^a	ACT	CAC	CAG
MTB-39	CAC	CAA	GAC	TTG^a	ACC^a	C	TAT	GGC	ATC^a	ACT	CAC	CAG
MTB-40	CGC^a	CAA	GAC	TCG	ACC^a	C	TAT	GGC	GTG^a	ACT	CAC	CAG
注：a. 表示突变型，其余为野生型

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