A multiplex PCR-based method to identify strongylid parasite larvae recovered from ovine faecal cultures and/or pasture samples
A multiplex PCR-based method to identify strongylid parasite larvae recovered from ovine faecal cultures and/or pasture samples
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DOI：
10.1016/j.vetpar.2013.12.002
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发表时间：
2014-02-01
影响因子：
2.6
通讯作者：
Bouchet, C. L. G.
中科院分区：
文献类型：
--
作者：
Bisset, S. A.;Knight, J. S.;Bouchet, C. L. G.
开发了一种基于多重PCR的方法，以克服显微镜检查的局限性，作为一种手段，从广泛的圆线虫寄生虫物种中常见的羊在混合羊-牛放牧的情况下，在新西兰识别个人感染性幼虫。该策略采用的目标是线虫核糖体DNA的第二个内转录间隔区（ITS-2）区域中独特的种特异性序列标记，并利用个体幼虫裂解物作为反应模板。基本试验涉及两组反应，旨在靶向新西兰条件下绵羊粪便培养物中最常遇到的10种圆线虫（即捻转血矛线虫、环状Teladorsagia circumcincta、阿氏毛圆线虫、蛇形毛圆线虫、镜状毛圆线虫、古柏线虫、瘤古柏线虫、斯氏线虫、绵羊夏贝尔线虫和细静脉食口线虫）。在每个反应中使用五种物种特异性引物以及一对“通用”（保守）引物。通常扩增两种产物，一种通过与物种无关的通用引物对（提供阳性PCR对照）扩增，另一种（其大小指示存在的物种）通过适当的物种特异性引物与一种或另一种通用引物组合扩增。如有必要，随后可使用专为检测绵羊粪便培养物中不常出现的菌种（即奥斯特线虫、细刺奥斯特线虫、点状古柏线虫、细颈线虫和三角头丘口线虫）而设计的引物检测未通过这些反应鉴别的任何幼虫。对分布在新西兰各地的16个不同农场的羔羊培养的>5500个线虫幼虫进行的测定结果表明，其中92.8%的线虫幼虫最初获得了阳性鉴定，而另外4.4%的反应产生了通用的但没有可见的特异性产物，2.8%的反应没有产生可辨别的PCR产物（表明DNA模板不足或质量差）。在仅产生通用产品的反应中，91%在测定重新运行中得到阳性鉴定，导致含有可重复模板的反应的失败率仅与0.4%相似。虽然该方法的开发主要是为了提供一种可靠的方法来识别个体圆线虫幼虫的下游分子应用，但它可能具有各种其他研究和实际应用，这些研究和实际应用目前使用其他方法不容易实现。(C)2013爱思唯尔有限公司版权所有。
A multiplex PCR-based method was developed to overcome the limitations of microscopic examination as a means of identifying individual infective larvae from the wide range of strongylid parasite species commonly encountered in sheep in mixed sheep-cattle grazing situations in New Zealand. The strategy employed targets unique species-specific sequence markers in the second internal transcribed spacer (ITS-2) region of ribosomal DNA of the nematodes and utilises individual larval lysates as reaction templates. The basic assay involves two sets of reactions designed to target the ten strongylid species most often encountered in ovine faecal cultures under New Zealand conditions (viz. Haemonchus contortus, Teladorsagia circumcincta, Trichostrongylus axei, Trichostrongylus colubriformis, Trichostrongylus vitrinus, Cooperia curticei, Cooperia oncophora, Nematodirus spathiger, Chabertia ovina, and Oesophagostomum venulosum). Five species-specific primers, together with a pair of "generic" (conserved) primers, are used in each of the reactions. Two products are generally amplified, one by the generic primer pair regardless of species (providing a positive PCR control) and the other (whose size is indicative of the species present) by the appropriate species-specific primer in combination with one or other of the generic primers. If necessary, any larvae not identified by these reactions can subsequently be tested using primers designed specifically to detect those species less frequently encountered in ovine faecal cultures (viz. Ostertagia ostertagi, Ostertagia leptospicularis, Cooperia punctata, Nematodirus filicollis, and Bunostomum trigonocephalum). Results of assays undertaken on >5500 nematode larvae cultured from lambs on 16 different farms distributed throughout New Zealand indicated that positive identifications were initially obtained for 92.8% of them, while a further 4.4% of reactions gave a generic but no visible specific product and 2.8% gave no discernible PCR products (indicative of insufficient or poor quality DNA template). Of the reactions which yielded only generic products, 91% gave positive identifications in an assay re-run, resulting in a failure rate of just similar to 0.4% for reactions containing amplifiable template. Although the method was developed primarily to provide a reliable way to identify individual strongylid larvae for downstream molecular applications, it potentially has a variety of other research and practical applications which are not readily achievable at present using other methods. (C) 2013 Elsevier B.V. All rights reserved.