环介导等温扩增联合横向流动试纸条检测简单异尖线虫/派氏异尖线虫方法的建立

A LOOP-MEDIATED ISOTHERMAL AMPLIFICATION TECHNIQUE COMBINED WITH A LATERAL FLOW DIPSTICK FOR THE DETECTION OF ANISAKIS SIMPLEX SENSU STRICTO/ANISAKIS PEGREFFII IN COMMERCIAL FISH

简单异尖线虫复合种(Anisakissimplexspeciescomplex)是人兽共患寄生虫,其中的简单异尖线虫(A. simplex sensu stricto)和派氏异尖线虫(A. pegreffii)能引起人异尖线虫病。本研究利用环介导等温扩增技术(loop-mediatedisothermalamplification,LAMP)结合流动试纸条(lateralflowdipstick,LFD)建立了简单异尖线虫/派氏异尖线虫的快检技术。本研究以简单异尖线虫核糖体DNA的内转录间隔区(ribosomal internal transcribed spacer 2, rDNA-ITS2)序列为检测靶标,设计6条特异性的引物进行(荧光)LAMP反应。将生物素化的LAMP产物与异硫氰酸荧光素标记的探针杂交并通过LFD可视化显示。结果表明,本研究建立的LAMP-LFD可以特异性检测出简单异尖线虫/派氏异尖线虫,而对其它10种蠕虫的检测结果呈阴性;针对两个异尖线虫姊妹种的第三期幼虫(third-stagelarvae,L3)的检测灵敏度为单条虫体基因组DNA的10–5倍。优化后的LAMP反应时间为40min,加之探针杂交和LFD显色,整个检测时程只需50min。利用本LAMP-LFD方法对2015—2017年收集于140尾海鱼的1573条线虫进行检测的结果表明,简单异尖线虫/派氏异尖线虫是东海海域的优势种,这与经rDNA-ITS1-5.8S-ITS2测序的结果一致。因此, LAMP-LFD方法能快速、灵敏且准确地检测简单异尖线虫/派氏异尖线,在经济鱼类中简单异尖线虫复合种的筛检和常规检验检疫具有巨大潜力。

Anisakis simplex sensu stricto (s.s.) and Anisakis pegreffii are two sibling species of morphospecies A. simplex, which are fish borne zoonotic parasites dominantly responsible for human anisakiasis. A novel loop-mediated isothermal amplification (LAMP) combined with a lateral flow dipstick (LFD) was developed for the visual detection of A. simplex s.s./A. pegreffii (named LAMP-LFD) in commercial fish. Six primers that specifically recognized the conserved region of ribosomal internal transcribed spacer 2 sequence (rDNA-ITS2) of A. simplex s.s./A. pegreffii were designed, and the (fluorescent) LAMP assays were performed. The LAMP products were biotinylated and hybridized with a fluorescein isothiocyanate-labelled probe and visualized for 3-5min by trapping at LFD test line. The results showed that the LAMP-LFD method could specifically detect A. simplex s.s./A. pegreffii from other 10 worm species through a 40-min nucleic acid amplification at 63°C. The detection limit was 6×10^3copies/μL of recombinant rDNA-ITS2 of A. simplex s.s./A. pegreffii and the equivalent of 10^-5 single third-stage larvae of both A. simplex s.s. and A. pegreffii. The epidemiological investigation by the LAMP-LFD method was accord with that of the DNA sequencing of rDNA-ITS1-5.8S-ITS2, which indicated that A. pegreffii are dominant Anisakis species of marine fish in the East China Sea. Therefore, the LAMP-LFD method enriches in rapidity, sensitivity, and accuracy and has great potential in the routine detection and quarantine of A. simplex s.s./A. pegreffii in commercial fish.