合成PCR和LAMP引物,分别扩增体外纯培养的阴道毛滴虫虫体DNA,建立阴道毛滴虫PCR和LAMP检测方法。通过检测临床镜检阴道毛滴虫阳性和阴性的阴道分泌物样品各40份,比较两种方法在临床应用的可行性;通过扩增解脲支原体、蓝氏贾第鞭毛虫、金黄色葡萄球菌、白假丝酵母菌、大肠埃希菌等的DNA,评价2种方法的特异性;测定体外纯培养的阴道毛滴虫虫体DNA浓度,经倍比稀释后,评价2种方法的灵敏度。结果显示,阴道毛滴虫DNA扩增后,PCR方法可检测出263 bp特异条带;LAMP方法显示扩增体系颜色由红色变为黄色,琼脂糖凝胶电泳可见DNA梯状条带。可行性检测结果显示,PCR检测40份临床阴道毛滴虫阳性样品和40份阴性样品的符合率分别为100%和100%,LAMP方法的符合率分别为100%和97.5%。特异性检测结果显示,2种方法均无法扩增其他5种病原体的DNA。灵敏度检测结果显示,PCR和LAMP方法的检测下限分别为0.5006 ng/μl和0.05006 ng/μl。提示PCR和LAMP技术都可用于阴道毛滴虫的检测,两种方法的特异性都较高,LAMP方法的灵敏度优于PCR方法。

To establish PCR and LAMP method for detection of Trichomonas vaginalis, the primers were synthesized, and the DNA of cultured of T. vaginalis was amplified. The feasibility of the two methods in clinical application was compared by detecting 40 T. vaginalis-positive and 40 T. vaginalis-negative samples of vaginal discharge identified by clinical microscopy. The specificity of the two methods was analyzed by amplifying DNA of Mycoplasma urealytium, Giardia lamblia, Staphylococcus aureus, Candida albicans and Escherichia coli. The sensitivity of the two methods was analyzed by determining the concentration of cultured T. vaginalis DNA after serial dilution. The results showed that PCR amplification of T. vaginalis DNA resulted in a 263-bp specific band, and the LAMP method caused a change of amplification product color from red to yellow, presenting the DNA ladder on agarose gel electrophoresis. The feasibility results showed that the coincidence rate of the 40 positive and 40 negative clinical samples by PCR were 100% and 100%, while LAMP method resulted in 100% and 97.5%, respectively. The specificity results showed that the DNA of 5 other pathogens could not be amplified by the two methods. The sensitivity test showed that the lower detection limit of the PCR method was 0.5006 ng/μl and the LAMP method 0.05006 ng/μl. These results indicate that the both methods are highly specific and the LAMP is superior to PCR in sensitivity.