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

发布时间：2018-03-03 04:34

本文选题：内弯宫脂线虫　切入点：环介导等温扩增　出处：《中国预防兽医学报》2017年02期 　论文类型：期刊论文

【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。
[Abstract]:In order to establish a rapid LAMP-LFD detection technique for H. aduncumum, the method of loop mediated isothermal amplification (Lamp) and lateral flow test strip (LFDs) was combined in this study. Three pairs of specific primers (including upstream primers labeled by biotin) were designed to detect the internal transcriptional spacer (DNA(r) sequence of nematodes. The product was hybridized with a probe labeled with fluorescein isothiocyanate (FITC). The results on LFD showed that the LAMP-LFD method could specifically detect the nematode. The detection sensitivity of the recombinant plasmid containing r DNA-ITS2 sequence was 1.4 脳 10 ~ 2 copies / 渭 L, which was 100 times higher than that of the conventional PCR method. The third stage larva of Dioscorea innervalis, The detection sensitivity of single worm was 10-6 times as high as that of genomic DNA of single worm. The detection time course of optimized LAMP was only 40 mins. The detection results of real samples showed that the results obtained by LAMP-LFD method were consistent with those obtained by PCR binding sequencing. LAMP-LFD can be used to detect the nematodes specifically, sensitively and efficiently, and has a good application prospect in the inspection and quarantine of the nematode.
【作者单位】：宁波大学生物技术系;舟山出入境检验检疫局;
【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)
【分类号】：S852.7

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