CAJ | 학술논문

目的建立特异、灵敏、高效的TaqMan-MGB荧光定量PCR方法用于快速检测肠聚集性大肠埃希菌(EAEC)aggR基因.方法试验选用0.16、0.24、0.32、0.40、0.48和0.56 μmol/L引物和探针终浓度,采用矩阵法优选引物和探针的最佳浓度.根据GenBank上公布的EAEC特异基因aggR序列,在其保守区域设计引物与TaqMan-MGB探针,并对荧光定量PCR体系与反应条件进行优化,同时验证方法的特异度、敏感度和重复性.结果采用0.48 μmol/L的引物和0.24 μmol/L的探针终浓度,获得的ct值最小而△Rn值最大,59.0℃为该反应体系的最佳退火温度.该法对EAEC aggR基因检测具有高度特异性,与肠致病性大肠埃希菌、肠产毒性大肠埃希菌、肠侵袭性大肠埃希菌、肠出血性大肠埃希菌、大肠埃希菌、小肠结肠炎耶尔森菌、甲型副伤寒沙门菌、鼠伤寒沙门菌、弗氏志贺菌、宋内志贺菌、单核细胞增生李斯特菌、铜绿假单胞菌、奇异变形杆菌、蜡样芽孢杆菌、金黄色葡萄球菌、副溶血性弧菌等均无交叉反应,且方法灵敏度高、重复性好,最低检出限可达到6.5×100拷贝/μL(或13拷贝/反应体系),整个操作过程仅需2h.结论本研究建立的TaqMan-MGB荧光定量PCR方法特异、灵敏、高效,适用于EAEC临床病例的快速检测和食物中毒等突发事件的应急诊断.
목적 건립특이、령민、고효적TaqMan-MGB형광정량PCR방법용우쾌속검측장취집성대장애희균(EAEC)aggR기인.방법 시험선용0.16、0.24、0.32、0.40、0.48화0.56 μmol/L인물화탐침종농도,채용구진법우선인물화탐침적최가농도.근거GenBank상공포적EAEC특이기인aggR서렬,재기보수구역설계인물여TaqMan-MGB탐침,병대형광정량PCR체계여반응조건진행우화,동시험증방법적특이도、민감도화중복성.결과 채용0.48 μmol/L적인물화0.24 μmol/L적탐침종농도,획득적ct치최소이△Rn치최대,59.0℃위해반응체계적최가퇴화온도.해법대EAEC aggR기인검측구유고도특이성,여장치병성대장애희균、장산독성대장애희균、장침습성대장애희균、장출혈성대장애희균、대장애희균、소장결장염야이삼균、갑형부상한사문균、서상한사문균、불씨지하균、송내지하균、단핵세포증생리사특균、동록가단포균、기이변형간균、사양아포간균、금황색포도구균、부용혈성호균등균무교차반응,차방법령민도고、중복성호,최저검출한가체도6.5×100고패/μL(혹13고패/반응체계),정개조작과정부수2h.결론 본연구건립적TaqMan-MGB형광정량PCR방법특이、령민、고효,괄용우EAEC림상병례적쾌속검측화식물중독등돌발사건적응급진단.
Objective To establish a TaqMan-MGB fluorescence quantitative PCR method for rapid detection of aggR gene in enteroaggregative Escherichia coli (EAEC).Methods Primers and probes at final concentrations of 0.16,0.24,0.32,0.40,0.48 and 0.56 μmol/L were selected to detemine the optimal concentration by matrix method.According to EAEC aggR gene sequence in GenBank,the primer and TaqManMGB probe were designed in the conserved region,and fluorescence quantitative PCR system and reaction conditions were also optimized.The specificity,sensitivity and reproducibility of this method were verified.Results Primer and probe at final concentrations of 0.48 μmol/L and 0.24 μmol/L respectively were selected for the method obtaining minimum ct value and maximum △Rn value.The optimal annealing temperature for the reaction system was 59.0 ℃.This method was highly specific for EAEC aggR genetic test and had no crossreaction with enteropathogenic E.coli,enterotoxingenic E.coli,enteroinvasive E.coli,enterohaemorrhagic E.coli,E.coli,Y.enterocolitica,S.paratyphi,S.typhimurium,S.flexneri,S.sonnei,L.monocytogenes,P.aeruginosa,P.mirabilis,B.cereus,S.aureus and V.parahaemolyticus.This method had high sensitivity and good reproducibility,with the detection limit of 6.5 ×100 copies/μL (or 13 copies/reaction system).The whole operation took only 2h.Conclusions The TaqMan-MGB fluorescence quantitative PCR assay is a quick,sensitive and specific method for rapid detection of EAEC cases and emergency diagnosis of food poisoning.