CAJ | 학술논문

目的利用多重荧光定量PCR技术,建立一种快速、准确、特异检测肠出血性大肠杆菌O157:H7的定量方法.方法选取肠出血性大肠杆菌O157:H7编码脂多糖基因(rfbE)和编码鞭毛抗原基因(fliC)作为检测的靶基因,设计引物和TaqMan-MGB探针,探针的5'端分别用FAM和HEX进行荧光标记,3'端标记MGB.优化PCR扩增体系,对多重实时荧光定量PCR方法的特异性、灵敏度、重复性评价,同时进行一定数量临床样本鉴定,与常规方法进行比较.结果本研究所建立的多重实时荧光定量PCR方法可准确、特异地检测和鉴定肠出血性大肠杆菌O157:H7,能够有效甄别肠出血性大肠杆菌O157:H7与非H7菌株,其他菌株均无阳性结果;该方法的灵敏度可达到10 CFU/ml;定量检测的批间和批内变异系数均小于5%;对66例临床样本进行评价,结果显示15例肠出血性大肠杆菌O157:H7阳性,2例为肠出血性大肠杆菌O157:非H7阳性,其中16例与常规培养法结果符合,符合率达到98.49%.结论本研究建立的检测肠出血性大肠杆菌O157:H7多重实时荧光定量PCR方法快速,结果准确、可靠,操作简便,为肠出血性大肠杆菌O157:H7的临床诊断、现场流行病学调查和食品安全监测提供了新的鉴定方法.
목적 이용다중형광정량PCR기술,건립일충쾌속、준학、특이검측장출혈성대장간균O157:H7적정량방법.방법 선취장출혈성대장간균O157:H7편마지다당기인(rfbE)화편마편모항원기인(fliC)작위검측적파기인,설계인물화TaqMan-MGB탐침,탐침적5'단분별용FAM화HEX진행형광표기,3'단표기MGB.우화PCR확증체계,대다중실시형광정량PCR방법적특이성、령민도、중복성평개,동시진행일정수량림상양본감정,여상규방법진행비교.결과 본연구소건립적다중실시형광정량PCR방법가준학、특이지검측화감정장출혈성대장간균O157:H7,능구유효견별장출혈성대장간균O157:H7여비H7균주,기타균주균무양성결과;해방법적령민도가체도10 CFU/ml;정량검측적비간화비내변이계수균소우5%;대66례림상양본진행평개,결과현시15례장출혈성대장간균O157:H7양성,2례위장출혈성대장간균O157:비H7양성,기중16례여상규배양법결과부합,부합솔체도98.49%.결론 본연구건립적검측장출혈성대장간균O157:H7다중실시형광정량PCR방법쾌속,결과준학、가고,조작간편,위장출혈성대장간균O157:H7적림상진단、현장류행병학조사화식품안전감측제공료신적감정방법.
Objective To develop a rapid, sensitive and specific assay based on multiplex real-time PCR for detecting and identifying Escherichia coli O157: H7. Methods The lipopolysaccharide gene (rJbE) and H7 flagellar antigen gene(fliC) of Escherichia coli O157:H7 was chosen as targets, and then the primers and TaqMan-MGB probe were designed. The 5'end of probes was labeled with FAM and HEX fluo-resceins respectively; the 3'end of probes was labeled with MGB. The PCR reaction was optimized systemati-cally. Then the specificity, sensitivity and reproducibility of multiplex real-time PCR were estimated. Final-ly, multiplex real-time PCR was applied to detected clinical specimens. Results Escherichia coil O157:H7 were detected by multiplex real-time PCR accurately and quickly, which could distinguish Escherichia coli O157:H7 from O157: non-H7. Meanwhile, none of other bacteria could be identified. The sensitivity was 10 CFU/ml in pure culture. The coefficient of variation of intra-assay and inter-assay was less than 5%. When this assay was applied directly to identify 66 clinical specimens, the results showed that t5 were positive to Escherichia coil O157:H7 and 2 were positive to Escherichia coil O157: non-H7, in which 16 was the same to the results obtained from the conventional assays. The coincidence was 98.49%. Conclusion It is showed that multiplex real-time PCR is a reliable, accurate and feasible assay for detecting and identifying Escherich-ia coli Oi57: H7, The assay reported here provided a tool for analysis and diagnosis in the field of detecting clinical pathogens, epidemiologic survey and food safety monitoring.