内弯宫脂线虫环介导等温扩增联合横向流动试纸条检测方法的建立  

Rapid detection of Hysterothylacium aduncum by loop-mediated isothermal amplification combined with lateral flow dipstick

作　　者：乔艳[1] 周前进[1] 李孝军 陈炯[1] QIAO Yan ZHOU Qian-Jin LI Xiao-Jun CHEN Jiong(Department of Biological Science ＆ Technology, Ningbo University, Ningbo 315211, China Zhoushan Entry-Exit Inspection and Quarantine Bureau, Zhoushan 316000, China)

机构地区：[1]宁波大学生物技术系,浙江宁波315211 [2]舟山出入境检验检疫局,浙江舟山316000

出　　处：《中国预防兽医学报》2017年第2期137-142,共6页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家高技术研究发展计划(863计划)(2012AA020101);浙江省自然科学基金项目(LY14C180002);浙江省科技计划项目(2012C32016);宁波市科技创新团队项目(2015C110018);宁波市科技富民项目(2016C10042)

摘　　要：为建立内弯宫脂线虫(H.aduncum)的LAMP-LFD快速检测技术,本研究将环介导等温扩增技术(LAMP)与横向流动试纸条(LFD)的检测方法结合,以内弯宫脂线虫核糖体DNA(r DNA)的内转录间隔区(ITS)序列为检测靶标,设计了3对特异性引物(其中上游内引物由生物素标记)进行LAMP扩增反应,产物与经异硫氰酸荧光素(FITC)标记的探针杂交,在LFD上完成结果显示。结果表明,LAMP-LFD方法能够特异性地检测内弯宫脂线虫,对简单异尖线虫等8种其它常见鱼类寄生蠕虫检测呈阴性。对含有内弯宫脂线虫r DNA-ITS2序列的重组质粒的检测灵敏度为1.4×10~2拷贝/μL,为常规PCR检测方法的100倍;对内弯宫脂线虫第三期幼虫,单虫检测灵敏度为单条虫体基因组DNA的10-6倍。优化后LAMP检测时程仅40 min。对实际样品的检测结果表明,LAMP-LFD方法检测内弯宫脂线虫与PCR结合测序方法获得的结果一致。因此,利用LAMP-LFD能够特异、灵敏、高效地检测出内弯宫脂线虫,具有在该线虫的检验检疫中良好的应用前景。In the present study, a loop-mediated isothermal amplification （LAMP） method combined with a lateral flow dipstick （LFD） was developed for rapid detection Hysterothylacium aduncum. Three pairs of primers were designed for the usage of LAMP reaction, targeting the internal transcribed spacer 2 （ITS2） of ribosomal DNA （rDNA）. Among the primers, the forward inner primer （FIP） was biotinylated. Thereafter, the LAMP product was hybridized with a FITC-labeled probe and the hybrid was visualized by LFD. The results indicated that LAMP-LFD was able to specifically detect H.aduncum and no amplification was detected from the genomic DNA of other 8 common worms parasitizing fish. The detection limit was 1.4x10^2 copies/μLof recombinant plasmid carrying rDNA-ITS2 sequence of H.aduncum, which was 100 fold sensitive than that of the PCR assay. The assay was capable to detect 10.6 genomic DNA of single third stage juvenile. The whole procedure of the assay completed in 40 min, including 30 min for LAMP and 10 min for probe hybridization and visualization in LFD. Moreover, the positive rate of the LAMP-LFD assay for H.aduncumdetection was identical to the PCR assay for the clinical samples. Therefore, the LAMP-LFD israpid, sensitive and specific assay, which has the potential to be used in routine detection and quarantine of H.aduncum.

关 键 词：内弯宫脂线虫 环介导等温扩增 横向流动试纸条 检测 

分 类 号：S852.7[农业科学—基础兽医学]