CAJ | 학술논문

目的利用多重荧光定量PCR技术,建立一种快速、准确、特异甄别霍乱弧菌的定量方法.方法分别根据霍乱弧菌霍乱毒素A亚单位基因(ctxA)和糖基转移酶基因(LPSgt)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VIC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考核鉴定,与常规方法进行比较.结果该方法可准确、特异地鉴定霍乱弧菌,同时能够甄别O139群霍乱弧菌.全部的霍乱弧菌用ctxA基因对应引物和探针检测均为阳性,其中只有O139群霍乱弧菌出现LPSgt基因阳性,其他菌株均无阳性结果.检测的灵敏度为2×102 cfu/ml.同时对收集的45例临床样本进行鉴定,结果显示10例为霍乱弧菌,其中O139群有4例,其余均为阴性结果,与常规鉴定方法一致.结论研究建立的多重荧光定量PCR方法特异、灵敏、快速,可有效鉴定产毒霍乱弧菌及甄别O139群霍乱弧菌,可用于霍乱监测和防制工作中的实验室诊断.
목적 이용다중형광정량PCR기술,건립일충쾌속、준학、특이견별곽란호균적정량방법.방법 분별근거곽란호균곽란독소A아단위기인(ctxA)화당기전이매기인(LPSgt)작위검측적파기인,설계인물화TaqMan-MGB탐침,탐침적5'단분별용FAM화VIC진행형광표기,3'단표기MGB.우화PCR확증체계,대다중실시형광정량PCR방법적특이성、령민도、중복성평개,동시진행일정수량림상양본고핵감정,여상규방법진행비교.결과 해방법가준학、특이지감정곽란호균,동시능구견별O139군곽란호균.전부적곽란호균용ctxA기인대응인물화탐침검측균위양성,기중지유O139군곽란호균출현LPSgt기인양성,기타균주균무양성결과.검측적령민도위2×102 cfu/ml.동시대수집적45례림상양본진행감정,결과현시10례위곽란호균,기중O139군유4례,기여균위음성결과,여상규감정방법일치.결론 연구건립적다중형광정량PCR방법특이、령민、쾌속,가유효감정산독곽란호균급견별O139군곽란호균,가용우곽란감측화방제공작중적실험실진단.
Objective To develop a rapid, sensitive and specific assay method, based on multiplex real time PCR for identifying Vibrio cholerae and distinguishing Vibrio cholerae O139 serotype from Vibrio cholerae. Methods Cholera toxin A subunit gene (ctxA) and glycosyltransferase gene (LPSgt) were chosen as targets according to Vibrio cholerae and Vibrio cholerae O139 serotype,and then the primers and TaqMan-MGB probe were designed. The 5'end of probes was labeled with FAM and VIC fluoresceins respectively while the 3' end of probes was labeled with MGB. The PCR reaction was optimized systematically. Then the specificity, sensitivity and reproducibility of multiplex real time PCR were estimated. Finally, multiplex real time PCR was applied to detect the clinical specimens. Results Vibrio cholerae was identified by multiplex real time PCR accurately and quickly,which could distinguish Vibrio cholerae O139 serotype from Vibrio cholerae. Vibrio cholerae was identified positive for primer pairs and probes from ctxA gene, and Vibrio cholerae O139 serotype tested was positive for LPSgt gene. Meanwhile, none of other bacteria was identified. Sensitivity of the test was 2 × 102 cfu/ml. When this assay was applied directly to identify 45 clinical specimens, results showed that 10 were positive to Vibrio cholerae, in which 4 clinical specimens were positive to Vibrio cholerae O139 serotype. All the results were the same to the one that had been obtained from the conventional assays. Conclusion Our rsults showed that the multiplex real time PCR was a reliable,accurate and feasible method for identifying Vibrio cholerae that carrying toxin gene and distinguishing Vibrio cholerae O139 serotype from Vibrio cholerae. This method could be applied to the cholera surveillance, prevention and control system for identifying and distinguishing Vibrio cholerae in the labs.