我国东海沿海鱼类异尖科线虫RPA检测方法的建立

Development of Recombinase Polymerase Amplification Technique for Rapid Detection of Anisakidae in Coastal Fisheries in China

异尖科线虫病(Anisakiasis)是一种重要的食源性人兽共患线虫病,主要因食用生的或未煮熟的含异尖科线虫Ⅲ期活的幼虫的海鱼而引起。本研究利用重组酶聚合酶扩增技术(RPA)建立了异尖科线虫RPA的检测方法,首先对东海沿海舟山、温州、宁波、平潭、嘉兴市的九种鱼体内获得的线虫进行分离鉴定,获得了派氏异尖线虫、简单异尖线虫、典型异尖线虫、宫脂线虫和对盲囊线虫,然后根据获得的五种线虫的ITS区设计特异性引物。结果显示:本研究建立的RPA方法可特异性扩增出异尖科线虫340 bp左右的目的基因片段,可特异性检测出异尖科线虫,而对阔节裂头绦虫、华支睾吸虫、东方次睾吸虫、棘颚口线虫检测结果为阴性;优化后在35℃、25 min即可完成检测,其灵敏度可达1 pg/μL,人工污染实验结果显示呈阳性。此方法操作简单、便捷,对现场快速检测具有重要的意义。

Anisakiasidae nematode infection is an important food-borne zoonotic disease, mainly caused by eating raw or undercooked marine fishes containing its stage Ⅲ larvae. In this study, a detection method for Anisakidae infection was developed by using recombinant enzyme polymerase amplification technology(RPA). Firstly, the Anisakidae parasites were isolated from nine kinds of fishes in Zhoushan, Wenzhou, Ningbo, and Jiaxing cities in Zhejiang province and Pingtan in Fujian province along the East China Sea coast were and identified to be Anisakis peperii, Anisakis simplex, Anisakis typica and Hysterothylacium sp. and Contracaccum sp.Then, specific primers were designed according to their ITS regions. The results showed that the developed RPA method specifically amplified the target fragment of Anisakidae at about 340 bp but not Diphyllobothrium latum, Clonorchis sinensis, Orientia orientalis and Gnathostoma siamense. After optimization, the detection could be completed at 35℃ for 25 min and the limit was 1 pg/μL. The clinical samples were tested positive. The operation of this method was simple and convenient, which was of great significance for rapid on-site detection of Anisakiasidae nematode infection.