高分辨率熔解曲线分析法检测大肠埃希菌gyrA基因突变

doi:10.3969/j.issn.1673-8640.2015.11.018

摘要/Abstract

摘要： 目的

探讨高分辨率溶解曲线(HRM)分析法在检测大肠埃希菌gyrA基因突变中的可行性。

方法

收集临床非重复分离的大肠埃希菌70株,常规聚合酶链反应(PCR)扩增大肠埃希菌的gyrA基因,采用序列分析法检测gyrA基因的变异情况。设计特异性引物,采用荧光定量PCR扩增这70株大肠埃希菌株的gyrA基因,通过对扩增产物进行HRM分析和熔解温度(Tm)值测定确定gyrA基因变异情况。将检测结果与序列分析法检测结果进行比较。

结果

基因测序显示70株大肠埃希菌中有43株gyrA基因发生了突变,突变类型分为5型。HRM分析法可以准确区分野生株和突变株,准确率达98.6%(69/70),正确检测出了43株突变菌株中的42株,且对不同的突变类型准确分型。Tm分析显示野生株和突变株之间以及各种突变型之间有明显差异。

结论

HRM技术具有准确性高、简单、快速、成本低廉、高通量等优点,可以作为检测大肠埃希氏菌gyrA基因突变耐药机制的新选择。

关键词: 大肠埃希菌, gyrA基因, 高分辨率熔解曲线

Abstract: Objective

To investigate the feasibility of high-resolution melting (HRM) analysis to detect gyrA gene mutation in Escherichia coli.

Methods

A total of 70 isolates of Escherichia coli were collected, and gyrA gene was amplified with conventional polymerase chain reaction(PCR). Gene mutation was detected by sequence analysis. Specific primer was designed. The gyrA gene was amplified by fluorescence quantitation PCR. The mutation of gyrA gene was detected by amplifying product HRM curve and melting temperature (Tm) analysis. The result was compared with that of gene sequence analysis.

Results

gyrA gene mutation sequence analysis showed that 43 of 70 isolates occurred gyrA gene mutation. In these mutations, 5 types were found. HRM analysis can accurately distinguish between wild and mutant isolates. The detection accuracy was 98.6% (69/70).The 42 of 43 mutant isolates were correctly detected, and different type gene mutations can be correctly typed. Tm analysis also showed the difference of wild and mutant isolates.

Conclusions

HRM analysis is accurate, simple, rapid, cheap and high-throughput, and it and would be a novel choice in analyzing drug resistance mechanism of Escherichia coli.

Key words: Escherichia coli, gyrA gene, High-resolution melting

中图分类号:

Q503

茆海丰, 刘洪书. 高分辨率熔解曲线分析法检测大肠埃希菌gyrA基因突变[J]. 检验医学, 2015, 30(11): 1138-1142.

MAO Haifeng, LIU Hongshu. The detection of gyrA gene mutation in Escherichia coli by high-resolution melting analysis[J]. Laboratory Medicine, 2015, 30(11): 1138-1142.

图/表 6

图1 部分菌株gyrA基因普通PCR扩增电泳图

图2 发生多点突变位的gyrA基因测序图注:箭头示处为突变位点

表1 70株大肠埃希菌gyrA基因突变情况及各种突变型的Tm值

组别	株数	突变位点	DNA碱基改变	Tm(℃)
组别	株数	突变位点	DNA碱基改变	$x ?$	$x ?$ ±2s范围
野生组	27	—	—	83.84	83.71~83.86
Ⅰ组	24	83位TCG→TTG+87位GAC→AAC	C→T、G→A	82.56	82.47~82.66
Ⅱ组	11	83位TCG→TTG	C→T	83.32	83.20~83.46
Ⅲ组	4	83位TCG→TTG+85位GTT→GTC+91位CGT→CGC	T→C	84.81	84.74~84.88
Ⅳ组	2	83位TCG→TTG+85位GTT→GTC+ 87位GAC→AAC+91位CGT→CGC	G→A、T→C	84.08
Ⅴ组	2	85位GTT→GTC+91位CGT→CGC	T→C、T→C	85.32

表1 70株大肠埃希菌gyrA基因突变情况及各种突变型的Tm值

组别	株数	突变位点	DNA碱基改变	Tm(℃)
组别	株数	突变位点	DNA碱基改变	$x ?$	$x ?$ ±2s范围
野生组	27	—	—	83.84	83.71~83.86
Ⅰ组	24	83位TCG→TTG+87位GAC→AAC	C→T、G→A	82.56	82.47~82.66
Ⅱ组	11	83位TCG→TTG	C→T	83.32	83.20~83.46
Ⅲ组	4	83位TCG→TTG+85位GTT→GTC+91位CGT→CGC	T→C	84.81	84.74~84.88
Ⅳ组	2	83位TCG→TTG+85位GTT→GTC+ 87位GAC→AAC+91位CGT→CGC	G→A、T→C	84.08
Ⅴ组	2	85位GTT→GTC+91位CGT→CGC	T→C、T→C	85.32

图3 70株大肠埃希菌溶解曲线标准化图形

图4 70株大肠埃希菌溶解曲线标准化温度差异图形

图5 70株大肠埃希菌的Tm峰形图

参考文献 16

[1]	肖永红,沈萍,魏泽庆,等.Mohnarin 2011年度全国细菌耐药监测[J].中华医院感染学杂志,2012,22(22):4946-4952.
[2]	赵倩,汪雅萍,应春妹,等.质粒介导aac(6')-Ib基因检测与喹诺酮类耐药[J].检验医学,2013,28(3):199-202.
[3]	BEARDEN DT,DANZIGER LH.Mechanism of action of and resistance to quinolones[J].Pharmacotherapy,2001,21(10 Pt 2):224S-232S.
[4]	TRUONG QC,NGUYEN VAN JC,SHLAES D,et al.A novel,double mutation in DNA gyrase A of Escherichia coli conferring resistance to quinolone antibiotics[J].Antimicrob Agents Chemother,1997,41(1):85-90.
[5]	崔生辉,李景云,马越,等.大肠埃希菌中氟喹诺酮耐药机制功能分析[J].中华微生物学和免疫学杂志,2008,28(4):338-342.
[6]	赵旺胜,江淑芳,顾兵,等.南京地区鲍曼不动杆菌喹诺酮类药物耐药基因突变的研究[J].临床检验杂志,2007,25(2):90-92.
[7]	肖永红,王其南.聚合酶链反应RFLP与SCCP检测伤寒杆菌DNA旋转酶基因变异[J].中国抗生素杂志,2000,25(4):286-288.
[8]	蒋萍,陈恒屹,张坚磊,等.耐喹诺酮类大肠埃希菌耐药机制的研究[J].中华医院感染学杂志,2007,17(6):635-638.
[9]	刘文恩,陈腊梅,李艳明,等.在中国大陆首次检出CTX-M-15型超广谱β-内酰胺酶基因[J].中国医师杂志,2007,9(10):1311-1314.
[10]	SLINGER R, BELLFOY D, DESJARDINS M, et al.High-resolution melting assay for the detection of gyrA mutations causing quinolone resistance in Salmonella enterica serovars Typhi and Paratyphi[J].Diagnostic Microbiol Infecti Dis,2007,57(4):455-458.
[11]	NIWA H,HOBO S,ANZAI T.A nuclectide mutation associated with fluorquinolone resistance observed in gyrA of in vitro obtained Rhodococcus equi mutants[J].Vet Microbiol,2006,115(1-3):264-268.
[12]	REED GH, KENT JO, WITTWER CT.High-resolution DNA melting analysis for simple and efficient molecular diagnostics[J].Pharmacogenomics,2007,8(6):597-608.
[13]	李刚,赵燕,魏取好,等.高分辨熔解曲线法检测临床常见KPC基因亚型[J].中华检验医学杂志,2011,34(6):520-522.
[14]	CHENG JC, HUANG CL, LIN CC, et al.Rapid detection and identification of clinically important bacteria by high-resolution melting analysis after broad-range ribosomal RNA real-time PCR[J]. Clin Chem,2006,52(11):1997-2004.
[15]	ONG DC, YAM WC, SIU GK, et al.Rapid detection of rifampicin- and Isoniazid-resistant Mycobacterium tuberculosis by high-resolution melting analysis[J]. J Clin Microbiol,2010,48(4):1047-1054.
[16]	HJELMSØ MH, HANEN LH, BAELUM J, et al.High-resolution nelt analysis for rapid comparison of bacterial community compositions[J]. Appl Environ Microbiol,2014,80(12):3568-3575.