进境水产品中典型异尖线虫恒温实时荧光检测法的建立与应用

Establishment and Application of a Real-time Fluorescence Loop-mediated Isothermal Amplification Method for the Detection of Anisakis typical in Imported Aquatic Products

摘要:

本研究建立了一套检测进境水产品中典型异尖线虫DNA的恒温实时荧光环介导等温扩增（loop-mediated isothermal amplification，LAMP）方法，根据LAMP方法原理，针对典型异尖线虫ITS2区域设计三套引物，特异性识别靶标基因。对比验证了典型异尖线虫、简单异尖线虫、短棘异尖线虫、抹香鲸异尖线虫、Anisakis nascettii、宫脂线虫、对盲囊线虫和颚口线虫的引物特异性；研究了模板浓度1 ng/μL至10 ag/μL的LAMP反应灵敏度，比传统的PCR方法高100倍；并在最低检测限1 fg/μL的质粒模板进行15次重复试验；对来自不同热带国家和地区的进境的41个实际样品进行测试，与传统PCR方法进行比较，假阳性率为0。恒温实时荧光快速检测方法适用于特异性检测典型异尖线虫。

Abstract:

The present study established a real-time fluorescence loop-mediated isothermal amplification (LAMP) method for detecting the Anisakis typical in imported aquatic products. According to the mechanism of LAMP, three sets of specific primers for targeting the ITS2 rDNA were designed for specific identification of target genes . The specificity of primers were tested among Anisakis typical, Anisakis simplex, Anisakis brevispiculata, Anisakis physeteris, Anisakis nascettii, Hysterothylacium Spp., Contracaccum Spp. and Gnathostoma spp.. The sensitivity of this LAMP method was tested with the template concentrations ranging from 1 ng/μL to 10 ag/μL, which was 100 times higher than the traditional PCR method. Besides, 15 times repeats were conducted at the detection limit of 1 fg/μL plasmid template. This LAMP method was used to detect Anisakis typical in 41 samples imported from different tropic countries and areas, and its false positive rate was zero compared with the conventional PCR method. The established real-time fluorescence LAMP method is suitable for specific A. typical detection in imported aquatic products.