论文部分内容阅读
为满足贝类寄生虫快速检测需求,本研究根据GenBank中发布的派琴虫ITS序列和包纳米虫18SrRNA序列的保守区域分别设计LAMP扩增引物,分别建立了两种寄生虫的单重LAMP检测方法,进一步对反应体系进行优化,组装成两种寄生虫的双重LAMP方法,同时还进行了特异性检测和敏感性检测,并通过对扩增产物进行限制性内切酶酶切检验双重LAMP方法的准确性。结果表明本研究建立的贝类寄生虫双重LAMP方法具有较好的特异性,检测派琴虫时的最低检测限为10拷贝/μL质粒DNA,检测包纳米虫时的最低检测限为100拷贝/μL质粒DNA,同时酶切试验也验证了双重LAMP检测结果的准确性。对随机采自东海和黄海的20份菲律宾蛤仔和10份美国进口牡蛎的检测结果显示,该双重LAMP方法在口岸检疫工作中可以作为派琴虫和包纳米虫的快速检测方法。
In order to meet the demand of rapid detection of shellfish parasites, LAMP amplification primers were designed according to the ITS sequences of sentinel insects and the conserved regions of 18SrRNA sequences of nano-packs published in GenBank, respectively. Single LAMP detection of the two parasites Method, the reaction system was further optimized and assembled into a dual LAMP method for both parasites. In addition, specific and sensitive assays were also performed and restriction endonuclease digestion was performed on the amplified product to detect double LAMP Accuracy The results showed that the double LAMP method of shellfish parasite established in this study had good specificity. The lowest detection limit was 10 copies / μL of plasmid DNA for detection of fusarium and the lowest detection limit was 100 copies / μL plasmid DNA, while digestion test also verified the accuracy of double LAMP test results. The test results of 20 Ruditapes philippinarum and 10 U.S. imported oysters randomly collected from the East China Sea and the Yellow Sea show that this dual LAMP method can be used as a rapid detection method for C. pyrenoidosa and M. burgdorferi in port quarantine work.