捻转血矛线虫PCR检测方法的建立及其应用  

Establishment of PCR Method for Detection of Haemonchus contortus and Its Application

作　　者：邹敏 要慧中 杨冰可 周璐露 赵光明[2] 杨文欢[2] 吴昌 林青[1,4] ZOU Min;YAO Huizhong;YANG Bingke;ZHOU Lulu;ZHAO Guangming;YANG Wenhuan;WU Chang;LIN Qing(College of Veterinary Medicine,Northwest A&F University,Yangling Shaanxi 712100,China;Shaanxi Animal Disease Prevention and Control Center,Xi’an 710299,China;Shangnan County Agricultural Comprehensive Law Enforcement Team,Shangnan Shaanxi 726300,China;State Key Laboratory of Veterinary Etiological Biology,Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China)

机构地区：[1]西北农林科技大学动物医学院,陕西杨凌712100 [2]陕西省动物疫病预防控制中心,西安710299 [3]商南县农业综合执法大队,陕西商南726300 [4]家畜疫病病原生物学国家重点实验室,中国农业科学院兰州兽医研究所,兰州730046

出　　处：《西北农业学报》2023年第1期139-144,共6页Acta Agriculturae Boreali-occidentalis Sinica

基　　金：陕西省重点研发计划项目(2020NY-017);陕西省农业农村厅省级农业专项资金项目(畜牧专项资金-XN14);西北农林科技大学大学生创新创业训练计划项目(X202010712098);家畜疫病病原生物学国家重点实验室(中国农业科学院兰州兽医研究所)开放课题资助项目(SKLVEB2019KFKT007)。

摘　　要：为了能从粪便中对捻转血矛线虫卵进行特异性检测,基于捻转血矛线虫ITS2-28S基因序列设计特异性引物,通过对退火温度反应条件优化,建立捻转血矛线虫特异性PCR检测方法。结果显示,该方法成功从捻转血矛线虫卵中扩增出约270 bp的特异性片段,其最低检出质量浓度为0.298 pg/μL,敏感性较高;进一步对捻转血矛线虫、细颈线虫、粗纹食道口线虫等多种线虫的虫卵DNA样品进行扩增,该方法仅能特异性扩增出捻转血矛线虫卵的目的片段,证明特异性良好。利用所建立的PCR检测方法对120只山羊的粪便样品进行检测,显示受检样品中捻转血矛线虫阳性率为38.3%,高于显微镜检测结果(阳性率为24.2%)。结果表明,该研究建立的PCR方法能够应用于临床检测山羊粪便样品中的捻转血矛线虫卵,可为山羊捻转血矛线虫病的诊断与防治提供有效技术支持。To detect the specificity of Haemonchus contortus eggs from feces,the specific primer was designed based on Haemonchus contortus ITS2-28S gene sequence,and the PCR method for detection of H.contortus eggs was established by opitimizing the reaction conditions of annealing temperature in this study.The results showed that the the PCR method successfully amplified a specific fragment of about 270 bp from the eggs of H.contortus,with a minimum detection concentration of 0.298 pg/μL and high sensitivity.DNA samples of eggs of various nematodes,such as H.contortus,Nematodirus spp.and Oesophagostomum asperum,were further amplified,and results proved that this method could only specifically amplify the target fragment of H.contortus eggs,indicating a good specificity.The established PCR method was further used to detect 120 samples of goat feces.The results revealed that the positive rate of H.contortus was 38.3%,which was higher than that using microscopy(24.2%);the PCR method established in this study could be applied to clinically detect the eggs of H.contortus in goat feces samples.This study provides an effective technical support for the diagnosis and control of H.contortus disease in goats.

关 键 词：山羊 捻转血矛线虫 聚合酶链反应 ITS2-28S基因 虫卵检测 

分 类 号：S852.731[农业科学—基础兽医学]