TaqMan-MGB探针实时荧光定量PCR联合检测难辨梭菌菌属基因及毒素基因方法学的建立

邵冬华; 季娜; 梁国威; 刘静

Abstract

邵冬华,季娜,梁国威,刘静.TaqMan-MGB探针实时荧光定量PCR联合检测难辨梭菌菌属基因及毒素基因方法学的建立[J].Chinese journal of Epidemiology,2014,35(5):576-580

TaqMan-MGB探针实时荧光定量PCR联合检测难辨梭菌菌属基因及毒素基因方法学的建立

Using the real-time PCR assay to establish TaqMan-MGB probe for rapid identification of Clostridium difficile and its toxin

Received:November 18, 2013

DOI：10.3760/cma.j.issn.0254-6450.2014.05.024

KeyWord: 难辨梭菌 TaqMan-MGB探针实时荧光定量PCR 菌属基因毒素基因

English Key Word: Clostridium difficile TaqMan-MGB probe Real-time PCR Bacteria genus gene Toxin gene

FundProject:

Author Name	Affiliation	E-mail
Shao Donghua	Department ofClinical Laboratory, Aerospace Center Hospital, Beijing 100049, China
Ji Na	Department ofClinical Laboratory, Aerospace Center Hospital, Beijing 100049, China
Liang Guowei	Department ofClinical Laboratory, Aerospace Center Hospital, Beijing 100049, China	htyysdh2006@qq.com
Liu Jing	Department ofClinical Laboratory, Aerospace Center Hospital, Beijing 100049, China

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Abstract:

目的建立一种简便、快速难辨梭菌菌属鉴定及其毒素基因筛查的荧光定量PCR检测方法。方法采用TaqMan-MGB探针，通过real-time PCR分析系统，同时检测难辨梭菌菌属基因磷酸丙糖异构酶（Tpi）、A毒素（TcdA）、B毒素（TcdB）及缺失部分基因的A毒素（TcdAT），从特异度、灵敏度及其抗干扰性等方面评价该方法，并联合全自动酶联荧光免疫系统（VIDAS）检测50 例临床不明原因腹泻病例粪便标本探讨其应用价值。结果难辨梭菌非产毒株 Tpi 基因（tpi）的检测下限是6×10^-2 CFU/μl，产毒株 tpi、tcdA、tcdB、tcdAT 的检测下限为 6×10^-1 CFU/μl；难辨梭菌非产毒株tpi检测下限批内、批间变异率分别为2.1%和2.3%；产毒株tpi、tcdA、tcdB、tcdAT的检测下限批内、批间变异率依次为3.0%和3.4%、2.9%和3.2%、5.3%和5.7%、2.7%和2.8%。临床常见分离菌株及梭菌属其他细菌对检测无干扰。50 例不明原因腹泻病例粪便标本中，采用TaqMan-MGB 探针实时荧光 PCR 与 VIDAS 酶标免疫检测 39 例阴性标本其符合率为 100%；6 例两方法检测均为阳性；3例VIDAS酶标免疫检测为可疑及2例为阴性，经TaqMan-MGB探针实时荧光PCR检测为A-/B＋菌株。结论建立的TaqMan-MGB探针实时荧光定量PCR具有高通量、高灵敏度和重复性好的特性，且可筛查携带缺失部分基因的TcdA难辨梭菌菌株。

English Abstract:

Objective To develop a real-time PCR assay for the rapid identification of Clostridium（C.） difficile and its toxin. Methods TaqMan real-time PCR was developed for the rapid identification of species specific gene（tpi）of C. difficile strains and the toxins A（TcdA），B（TcdB） and truncated toxin A（TcdAT）. Sensitivity，specificity and anti-interference ability of these methods were estimated，as well. Feces sampled from fifty diarrhea patients were tested by real-time PCR and compared to the results from VIDAS assay. Results The detection limits of tpi were 6×10^-2 CFU/μland 6 × 10^-1CFU/μ l in the non-oxin producing and toxin producing strains，respectively. The coefficients of variability（CV）of intra-assay and inter-assay for the detection limits of tpi in the non-toxin producing strain were 2.1% and 2.3% . The CVs of intra-assay and inter-assay for the detection limit of tpi，tcdA，tcdB and tcdAT in the toxin producing strain were 3.0% and 3.4%，2.9% and 3.2%，5.3% and 5.7%，2.7% and 2.8%，respectively. No interferance was detected from other genus or species in clostridium. From 50 clinical samples，thirty-nine of them were negative and six of them were positive under the TaqMan-MGB probe technique in accordance with VIDAS. Five samples appeared positive using the TaqMan-MGB probe technique，in which 3 were dubious and 2 were negative under VIDAS. Conclusion The newly developed method was a sensitive and reliable assay for rapid identification of C. difficile and its toxin. This method could be used to screen C. difficile isolates harboring truncated toxin A to avoid misdiagnosis，clinically.

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