猴源溶组织内阿米巴原虫巢式PCR检测方法的建立

Establishment of Nested PCR Assay for Detection of Monkey-Derived Entamoeba histolytica

目的:建立猴源溶组织内阿米巴原虫巢式PCR检测方法并初步应用于临床检测;方法:根据溶组织内阿米巴原虫16S r DNA基因序列,设计1对阿米巴属原虫保守引物和1对溶组织内阿米巴原虫特异性引物,建立猴源溶组织内阿米巴原虫巢式PCR检测方法,摸索出了该检测方法的最佳反应条件,进了行特异性和敏感性试验,并对96份猕猴粪便样品进行检测;结果:该巢式PCR方法能特异性地扩增出猴源溶组织内阿米巴原虫目的片段,与莫氏内阿米巴、波氏内阿米巴、迪斯帕内阿米巴、大肠内阿米巴、微小隐孢子虫等11种相关原虫基因组DNA无交叉反应;最低能检测到0.3 fg的阳性参照DNA;对临床样品检测结果表明该PCR技术检查阳性者显微镜检查均为阳性;结论:建立的巢式PCR方法具有高度的特异性和敏感性,对于溶组织内阿米巴原虫病的诊断和流行病学调查具有重要的应用价值。

Objective： To develop a nested PCR assay for the detection of monkey - derived Entamoeba histolytica.Method Based on the monkey - derived E. histolytica 16S rRNA gene sequences, one pair of Entamoeba genusspecific primers and one pair of E. histolytica species specific primers were designed. With these primers, a nestedPCR amplification conditions were optimized. A series of tests were conductedregarding thespecificity and sensi-tivity. Results： The nested PCR assay was specific and there is no cross - reaction with other parasites, such asE. moshkovskii,E, polecki,E, dispar,E, coli and C. parvum. The assay was able to detect as low as 0.3 fg ofcontrol positive DNA. The results of detection of clinical samples showed that the assay was coinciding with thetraditional method. Conclusion： The nested PCR assay reported here detects E. histolytica accurately and sensi-tively and will be an effective tool for molecular epidemiology and diagnosis of E. histolytica in monkey feces.