CAJ | 학술논문

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。
목적：건립일충간편、쾌속난변사균균속감정급기독소기인사사적형광정량PCR검측방법。방법채용TaqMan-MGB탐침，통과real-time PCR분석계통，동시검측난변사균균속기인린산병당이구매（Tpi）、A독소（TcdA）、B독소（TcdB）급결실부분기인적A독소（TcdAT），종특이도、령민도급기항간우성등방면평개해방법，병연합전자동매련형광면역계통（VIDAS）검측50례림상불명원인복사병례분편표본탐토기응용개치。결과난변사균비산독주Tpi기인（tpi）적검측하한시6×10-2 CFU/μl，산독주tpi、tcdA、tcdB、tcdAT적검측하한위6×10-1 CFU/μl；난변사균비산독주tpi검측하한비내、비간변이솔분별위2.1%화2.3%；산독주tpi、tcdA、tcdB、tcdAT적검측하한비내、비간변이솔의차위3.0%화3.4%、2.9%화3.2%、5.3%화5.7%、2.7%화2.8%。림상상견분리균주급사균속기타세균대검측무간우。50례불명원인복사병례분편표본중，채용TaqMan-MGB탐침실시형광PCR여VIDAS매표면역검측39례음성표본기부합솔위100%；6례량방법검측균위양성；3례VIDAS매표면역검측위가의급2례위음성，경TaqMan-MGB탐침실시형광PCR검측위A-/B＋균주。결론건립적TaqMan-MGB탐침실시형광정량PCR구유고통량、고령민도화중복성호적특성，차가사사휴대결실부분기인적TcdA난변사균균주。
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/μl and 6 × 10-1 CFU/μl 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.