Taq-Man实时荧光定量PCR同时检测O1和O139群霍乱弧菌

Simultaneous detection of Vibrio Cholerae O1 and O139 by real-time quantitative PCR

目的针对霍乱弧菌血清群各自编码O抗原的特异性基因建立能够快速、准确地同时检测O1和O139群霍乱弧菌的荧光PCR检测方法,并克服常用PCR检测方法中经常出现的假阳性现象。方法分别以rfbM和wbfR基因作为O1和O139群霍乱弧菌的模板设计引物和探针,通过优化反应条件建立了能够同时检测O1群和O139群霍乱弧菌的二联实时荧光定量PCR方法(FQ-PCR)。对该方法的特异性、灵敏度进行评估,并对10份模拟食品样品和133份临床样品进行了检测。结果特异性试验表明,该方法能选择性检测O1和O139群霍乱弧菌,能有效地避免O141群霍乱弧菌和拟态弧菌的假阳性,特异性为100%;灵敏度试验表明,O1群霍乱弧菌和O139群霍乱弧菌检出限分别为92cfu/ml和116cfu/ml;另外,试验建立的检测方法对模拟样品和临床样品检测结果与国标法的检测结果完全一致,符合率为100%。结论该实时荧光PCR检测方法能够同时检测O1和O139群霍乱弧菌,而且特异性好、灵敏度高,能有效克服假阳性,适用于基层疾病控制、口岸检疫单位日常疫情监测和临床诊断。

Objective Targeting the specific O-antigen gene cluster,a Taq-Man real-time fluorescence PCR assay was developed to detect O1 and O139 Vibrio cholera concurrently and avoid frequently occurred false positives.Methods Two pairs of specific primers and two Taq-Man fluorescent probes respectively targeting rfbM gene of Vibrio cholerae O1 and wbfR of O139 were designed.After optimization of conditions,the specialty and sensitivity of the detection method were evaluated and ten simulated food samples and 128 clinical samples were tested.Results The detection limits of Vibrio cholerae O1 and O139 were 92 CFU/ml and 116 CFU/ml,which were almost the same with the usual PCR method.The developed real-time fluorescence PCR protocol detected only Vibrio cholerae O1 and O139 and it was not affected by many normal food pathogens such as Staphylococcus aureus and Salmonella,especially Vibrio cholerae O141 which contained TCP genes and Vibrio mimicus which contained ZOT gene.The clinical detect results of the developed assay and routine culture method were exactly the same.Conclusion The developed detection assay could quantitatively detect Vibrio cholerae O1 and O139 concurrently in only 3 hours,and could avoid false positive,and thus was an efficacious method for detecting Vibrio cholerae O1 and O139 and could be used in a wide area such as entry-exit inspection and quarantine,food safety detection and clinical diagnose.