聚合酶链反应技术检测阴道毛滴虫的研究

Study on polymerase chain reaction for detection of Trichomonas vaginalis

目的:建立PCR检测阴道毛滴虫的方法。方法:按照滴虫TV-E650-1基因设计一对引物,采用PCR技术扩增滴虫目的基因,将扩增产物进行琼脂糖凝胶电泳,根据扩增及电泳结果判断PCR法体外诊断阴道毛滴虫感染的可行性。同时,通过与其它常见阴道微生物PCR结果对比,判断所设计引物的特异性;根据模板DNA的稀释倍数判断引物的敏感性。结果:设计的一对引物以滴虫DNA为模板,扩增出大小为438bp的基因片断,与目的基因相符;空白对照扩增阴性。以乳酸杆菌、大肠杆菌、阴道加德纳菌等阴道常见微生物DNA为模板,扩增结果均为阴性。将滴虫DNA稀释至1 000倍(相当于1.36pg/μL)仍可见明显扩增。结论:PCR检测滴虫方法可行,且准确,特异性好,敏感性高,能用于临床批量检测,可作为体外诊断阴道毛滴虫感染及流行病学调查的候选方法。

Objective：To establish a PCR method to detect Trichomonas vaginalis（T.vaginalis）.Methods：A pair of specific primers of TV-E650-1gene was designed.Determined the feasibility of PCR method in diagnosis of T.vaginalis infection according to the PCR amplification and electrophoresis results.At the same time,the specificity of the method was estimated by amplified DNA of other common vaginal microbes,and the sensitivity of the method is judged according to the dilution ratio of the template DNA as well.Results：A about 438 bp fragment had been detected when the PCR carried out by the template of T.vaginalis DNA,While none gene fragments were detected in blank control and in other group when PCR carried out by the template of other common vaginal microbes DNA,such as Lactobacillus,Escherichia coli and Vaginal Gardiner etc.Interestingly,the 438 bp gene fragment was still detected when the template DNA of T.vaginalis diluted to 1 000times（equivalent to 1.36pg/μL）.Conclusion：The method for detecting T.vaginalis infection by PCR is feasible,accurate,good specificity and highly sensitivity and it could be used for clinical diagnose in large quantities,and can also be a candidate method for screening of healthy population.