实时聚合酶链反应检测O1群和O139群霍乱弧菌方法的建立及应用

目的建立检测O1群和O139群霍乱弧菌的实时荧光聚合酶链反应（RT—PCR）方法并进行水体标本的检测评价。方法根据O1群和O139群霍乱弧菌O抗原编码基因rfb序列设计引物，利用SYBRGreen染料，建立同时检测霍乱弧菌O1群和O139群的RT—PCR方法，对所建立的方法分别进行实验室内的灵敏度、特异性及重复性评价。采集河口水标本增菌后进行RT—PCR检测，与分离培养方法比较评价实际应用价值。结果建立了检测O1群和O139群霍乱弧菌的双重RT—PCR方法，根据扩增产物的溶解温度能有效区分O1群和O139群霍乱弧菌两种目标片段的扩增；对其他10种弧菌染色体DNA没有扩增；RT—PCR检测524份河口水体标本的增菌液，与常规分离方法相比显示了明显的灵敏性，并且所有常规分离方法阳性标本其荧光PCR检测亦为阳性。结论以O1群和O139群rfb基因为目标检测片段建立的霍乱弧菌RT—PCR方法可用于环境水体样本中霍乱弧菌常规分离前的快速筛查。

Development and application of real-time polymerase chain reaction to detect Vibrio cholerae O1 and O139 in river water

Objective To develop a real-time SYBR Green polymerase chain reaction(PCR)for detection of Vibrio cholerae serogroups O1 and O139,and to evaluate its reliability through detection of estuary water samples.Methods Oantigen rfb genes specific for O1 and O139 were used for the design of PCR primers.The real-time SYBR Green PCR system in detecting O1 and O139 specific rfb genes in one tube was developed,and its sensitivity,specificity and reproducibility were evaluated.The ability of the real-time PCR in detection of estuary water samples was compared with the routine PCR and bacteria isolation.Results The amplification of O1 or O139 specific target gene could be detected according to the melt curve temperature of amplicons.No amplification was observed in the templates of other 10 non- cholerae vibrios.When comparing to the real-time PCR to bacteria isolation in detection of 524 estuary water samples,it showed high sensitivity,plus also positive in real-time PCR detection among all the samples in which bacteria of O1 or O139 were isolated.Conclusion The real-time SYBR Green PCR could be used as the first step of rapid environment screen of V.cholerae in water samples thus might enhance the efficiency of isolation in screening of large amount of water samples.