粪便样本溶组织内阿米巴、蓝氏贾第鞭毛虫双重TaqMan实时荧光聚合酶链反应检测方法的建立

Establishment of duplex TaqMan RT-PCR method for detection of Entamoeba histolytica and Giardia lamblia in fecal samples

目的探讨同时检测溶组织内阿米巴和蓝氏贾第鞭毛虫的双重TaqMan实时荧光聚合酶链反应(real-time fluorescent polymerase chain reaction,RT-PCR)方法。方法设计针对溶组织内阿米巴和蓝氏贾第鞭毛虫的引物对和探针,建立双重TaqMan RT-PCR的扩增体系,将PCR扩增产物序列导入pUC57质粒,测定方法的最低检测限,并通过临床粪便样本对该方法进行初步评价。结果本研究建立的双重TaqMan RT-PCR法对溶组织内阿米巴最低检测限为31.6 copies/μL,蓝氏贾第鞭毛虫最低检测限为32.0 copies/μL。总共纳入212例临床粪便样本,3例溶组织内阿米巴镜检阳性患者样本PCR扩增结果均为阳性,209例溶组织内阿米巴镜检阴性的患者样本1例PCR扩增结果阳性,其余为阴性。8例蓝氏贾第鞭毛虫涂片镜检阳性样本PCR扩增结果均阳性,204例蓝氏贾第鞭毛虫镜检阴性患者样本中1例PCR扩增结果为阳性,其余为阴性。通过扩增产物测序和Blast分析确定了镜检阴性PCR阳性患者样本中扩增序列属于目标病原体,且临床症状以及实验室检查结果也支持该诊断。其他致腹泻病原体PCR扩增结果阴性,不存在交叉反应。结论建立的双重TaqMan RT-PCR方法不仅可以检测出溶组织内阿米巴及蓝氏贾第鞭毛虫镜检阳性样本,还能检出两者镜检漏检的样本,敏感度高于镜检法。并且与其他腹泻病原体包括布氏嗜碘阿米巴、人芽囊原虫、邻单胞菌、气单胞菌、沙门氏菌、痢疾志贺菌菌株、球虫以及哈门氏内阿米巴不存在交叉反应,具有较好的特异性。

Objective To establish the duplex TaqMan RT-PCR method for detection of Entamoeba histolytica and Giardia lamblia in fecal samples.Methods Primer pairs and probes for Entamoeba histolytica and Giardia lamblia were designed and duplex TaqMan RT-PCR amplification system was constructed.PCR products were inserted into the pUC57 plasmid,and the lower limit of detection of the method was determined.Clinical stool samples were tested in order to evaluated the efficacy of the method.Results The detection limits of duplex TaqMan RT-PCR were 31.6 copies/µL for Entamoeba histolytica and 32 copies/µL for Giardia lamblia,respectively.Of the total of 212 clinical stool samples tested,all 3 samples with E.histolytica-positive patients by microscopy were positive by PCR,while 1 from 209 samples with E.histolytica-negative patients by microscopy were positive by PCR,and the remaining samples were negative.For Giardia lamblia,all 8 samples positive by microscopy were positive by PCR,and 1 from 204 sample with a microscopy-negative patient was positive by PCR,and the remaining samples were negative.The amplification product sequencing and blast analysis were used to confirm that the amplified sequence in the specimen of a patient with negative microscopy but positive PCR belongs to the targeted pathogen,supported by clinical symptoms and laboratory test results.PCR results for other diarrhea-causing pathogens were negative,indicating no cross-reactivity.Conclusions The dual TaqMan RT-PCR method developed in this study can not only detect microscopy-positive samples of Entamoeba histolytica and Giardia lamblia but also can detect samples that were missed by microscopy,with higher sensitivity than the microscopy method.Further,this detection method does not cross-react with other diarrhea-causing pathogens,including cross-react with other diarrhea-causing pathogens including Iodamoeba butschlii,Blastocystis hominis,Plesiomonas,Aeromonas,Salmonella,Shigella,Sphaerozoum fuscum,and Entamoeba hartmani,thus has a good specificity.