聚合酶链反应检测霍乱弧菌

Polymerase chain reaction for detection of vibrio cholerae and its clinical application

为快速、准确地检测霍乱弧菌，控制霍乱流行，研究建立了一种聚合酶链反应（ＰＣＲ）检测方法。根据霍乱弧菌肠毒素ＣＴ基因序列，自行设计一对特异引物，扩增片断为２９６ｂｐ，同时采用一种高效率、快速、简便的方法提取并纯化ＤＮＡ，该法能成功地检测各种样品中的产ＣＴ肠毒素Ｏ１群霍乱弧菌。应用ＰＣＲ技术和常规分离培养方法对９０９份腹泻患者粪便标本和１８份环境水样标本进行平行检测。结果表明，两种方法的检测结果一致，粪便标本阳性率为４．２％（３８／９０９）；水样标本为２２．２％（４／１８），其中１份粪便标本和１份水样标本，是经过第２次增菌培养后，检出用性，而ＰＣＲ方法未见假阳性及假阴性结果。表明ＰＣＲ方法准确、快速、灵敏，并可在２、３小时内检测出含３个菌细胞的标本。因此，该方法可广泛应用于检测各种临床、环境标本中的产ＣＴ肠毒素Ｏ１群霍乱弧菌。

n order to control and prevent cholera spreading,a polymerase chain reaction(PCR)method was established to detect the pathogen. According to the sequence of the cholera enterotoxin(CT)gene,we designed and synthesized a pair of speciffc primers to amplify 29 6 bp DNA fragments and we used a promising,rapid,and simple method to extract and purify DNA, Result showed:the PCR method was efficient to detect the CT gene operon of vibrio cholerae. 909 stcol samples taken from patients and 18 enviromental water samples were examined by PCR and broth cultures as well.The resuits are identical by using two methods:the positive rates are 4.2%(38/909) for stcol samples and 22.2%(4/18)for water smaples. Among them,one stool sample and One water sample were culture-positive only on the second time. Neither false positive nor false negative,result detected by PCR were observed.This method(PCR)was such Sensitive that the presence of 3 bacteria could be detetced within 2-3 hours. Thus,this method might be widely used to detect V. cholerae O1.