同时检测贝类派琴虫和包纳米虫的双重LAMP方法的建立  

Establishment of Duplex Loop-mediated Isothermal Amplification Method for Simultaneous Detection of Shellfish Infected with Perkinsus and Bonamia

作　　者：王彩霞[1] 冯春燕[1] 王巧黎 袁向芬[1] 邓俊花[1] 吕继洲[1] 吴绍强[1] 

机构地区：[1]中国检验检疫科学研究院动物检疫研究所,北京100029 [2]青岛市中心医疗集团,青岛266005

出　　处：《中国畜牧兽医》2015年第8期1935-1942,共8页China Animal Husbandry & Veterinary Medicine

基　　金：国家质检公益项目(201210018);国家科技支撑计划课题(2012BAK11B04)

摘　　要：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 establish a duplex loop-mediated isothermal amplification （LAMP） method for the detection of shellfish parasitic infection with Perkinsus and Bonamia, primers were designed according to the conserved region of ITS and 18S rRNA gene sequences of Perkinsus and Bonamia deposited in GenBank using the online Primer Explorer 4.0. Firstly, the respective single LAMP detection assay of two parasites were developed,and then the reaction conditions were op- timized by combining the two reaction systems into duplex LAMP method. The specificity and sensitivity of duplex LAMP method were examined and the confidence was evaluated by digesting the amplicons with respective endonuclease. The results showed that the established duplex LAMP method was specific enough to distinguish the two parasites from other parasites, and the detection limit of Perkinsus was 10 copies/μL plasmid DNA, the detection limit of Bonamia was 100 copies/μL plasmid DNA. The endonuclease digestion of duplex LAMP products certified the confidence of the established duplex LAMP method. The detection result of 20 Ruditales philip- pinarum samples collected randomly from the East China sea and Yellow sea and 10 Ostrea edulis samples imported from USA showed that the duplex LAMP method could be used as a rapid detection method for infection with Perkinsus and Bonamia.

关 键 词：贝类寄生虫病 环介导等温扩增反应(LAMP) 检测 派琴虫 包纳米虫 

分 类 号：Q78[生物学—分子生物学]