多重PCR鉴定三种颚口线虫方法的建立

DEVELOPMENT OF A MULTIPLEX PCR ASSAY TO IDENTIFY GNATHOSTOMA SPINIGERUM, GNATHOSTOA NIPPONICUM AND GNATHOSTOMA DOLORESI

为了建立快速、灵敏、可靠的鉴别颚口线虫虫种的方法,根据GenBank中发表的棘颚口线虫、日本颚口线虫和杜氏颚口线虫ITS-2序列,设计了3对特异引物,建立了这3种颚口线虫的单一PCR和多重PCR检测方法,并分别对单一PCR和多重PCR方法的特异性和敏感性进行了鉴定。结果显示单一PCR和多重PCR均能特异扩增出棘颚口线虫、日本颚口线虫和杜氏颚口线虫,其片段大小分别为282、358、183 bp,单一PCR对棘颚口线虫、日本颚口线虫和杜氏颚口线虫虫体DNA最小检出量分别为0.2、0.01、0.01 ng/μL。对宫脂线虫、异尖线虫、棘口吸虫以及迭宫绦虫均不能进行扩增。用建立的多重PCR方法对菲律宾、印度尼西亚、黑龙江颚口线虫等12条颚口线虫DNA模板进行扩增,经鉴定菲律宾与印度尼西亚的颚口线虫为棘颚口线虫,黑龙江的颚口线虫为日本颚口线虫。鉴定结果与原虫体样本的形态鉴定和测序分析结果一致。研究显示建立的多重PCR方法具有很强的特异性和较高的敏感性,可用于棘颚口线虫、日本颚口线虫和杜氏颚口线虫虫种的鉴定和流行病学调查。

Three pairs of primers specific for G.spinigerum(JN408326), G.nipponicum(JN408315) and G.doloresi(JN408329) were designed based on their ITS-2 sequences. Accordingly, single and multiplex PCR methods were developed to identify Gnathostoma species. The amplified products in both single and multiplex PCR methods were 282 bp for G.spinigerum, 358 bp for G.nipponicum and 183 bp for G.doloresi. The DNA detection limits were 0.2 ng for G.spinigerum, 0.01 ng for G.nipponicum and also 0.01 ng for G.doloresi. No PCR products were amplified from genomes of Hysterothylacium, Anisakis, Echinostoma and Spirometra erinaceieuropaei. In addition, 12 species of Gnathostoma isolated from Philippines, Indonesia and Heilongjiang were identified in multiplex PCR and the results were in agreement with morphology and sequence analysis. The isolates from Philippines and Indonesia were G.spinigerum and isolates from Heilongjiang were G. nipponicum. These data suggested that multiplex PCR methods could be used for identifying G. spinigerum, G. nipponicum and G. doloresi.