FTA-巢式PCR方法鉴定溶组织内阿米巴原虫的初步研究

目的建立一种简便,快速的FTA-巢式PCR方法用于鉴定粪便中溶组织内阿米巴原虫(Entamoeba histolytica, E.h).方法 收集门诊腹泻病人新鲜粪便,用光学显微镜进行初步检查.用FTA卡抽提镜检结果为阳性的粪便DNA,根据溶组织内阿米巴原虫的SSU -rRNA序列设计引物,进行巢式PCR扩增,对PCR产物进行琼脂糖凝胶分析,并对阳性产物进行测序和序列比对分析.结果 根据光学显微镜检测结果,挑选了44例镜检结果为阿米巴原虫阳性的腹泻病人粪便.经FTA-巢式PCR扩增,其中20例样本可扩增出427 bp左右的目的条带,目的条带的测序和序列分析结果表明为溶组织内阿米巴原虫.镜检方法与巢式PCR方法的阳性符合率为45.45%(20/44),将E.h与形态学相似的其他内阿米巴原虫进行了鉴定和区分.结论 本研究建立的FTA-巢式PCR方法具有简单,快速,准确等优点,为临床检验和流行病学调查中E.h的鉴别诊断提供了新的技术方法.

Identification of Entamoeba histolytica by FTA-nested PCR

To establish a simple and rapid FTA-nested PCR method for identification of the Entamoeba histolytica (E.h), the fecal samples of diarrheal patients were collected. The samples were examined with a microscope firstly, and 44 positive samples were selected. The genomic DNA of Entamoeba in positive samples was extracted by FTA filters. Primers were designed based on the SSU rRNA fragment of E.h and the plate DNA was amplified by nested PCR. All PCR products were detected by agarose gel electrophoresis, and then the target fragments were sequenced and analyzed by BLAST. The 427 bp fragment of DNA was detected from 20 fecal samples. Sequence analysis of 20 target fragments amplified by nested PCR showed that they were E.h. The consistent rate of microscopic examination and nested PCR was 45.45% (20/44). This nested PCR could identify and differentiate the pathogenic E.h from the non- pathogenic Entamoeba species which were morphologically identical (similar). It suggested that FTA-nested PCR is a simple, rapid and reliable technique for identifying E.h in human stool samples, and provided a new technical method for differential diagnosis of E.h in clinical examination and epidemiological survey.