牛羊肝片吸虫病实时荧光PCR检测方法的建立.pdf

2017年第 1期 Vo1．27No．1 检验检疫学刊 JOURNALOFINSPECTIONAND QUARANTINE 牛羊肝片吸虫病实时荧光PCR检测方法的建立 王彩霞 林祥梅 吴绍强 徐 赓 (1．中国检验检疫科学研究院 北京 100029；2．山东省沂南县畜牧兽 医局) 摘要 选择肝片吸虫保守的核糖体DNA ITS．2区域设计引物和TaqMan探针 ，通过对反应体系和反应 条件进行优化，建立 了实时荧光定量PCR检测肝片吸虫的方法。所构建方法检测质粒模板DNA 的动态范围 为 lxl0~-lxl0拷贝，敏感度可检测到 1拷贝质粒 DNA，而且与华支睾吸虫、姜片吸虫、卫氏并殖吸虫无交叉 反应，特异性 良好。以所建立的方法检测虫卵可以检测到2个肝片吸虫卵囊。结果表明．本研究所构建的牛羊 肝片吸虫病实时荧光PCR检测方法具有快速、灵敏和特异等优点．可满足养殖场和 口岸牛羊及其 肉制品肝 片吸虫的检测需要。 关键词 肝片吸虫；实时荧光PCR；敏感性；特异性；检测 中图分类号 Q789 EstablishmentofRealTimePCR M ethod forDetection ofFasciolosisin Bovineand Sheep WANG Caixia，LIN Xiangmei，WU Shaoqiang ，XU Geng2 (1．ChineseAcademyofInspectionandQuarantine，Beijing，100029； 2．BureauofAnimalHusbandryandVeterinaryinYinanCityofShandongProvince) Abstract：To facilitate rapid and sensitive diagnosisofFasciolosis，the realtime TaqM an PCR method wasdeveloped to detectFasciolosisinfection in bovine and sheep．Theprimersand Taqman probewere desinged and chosen to amplify the conserved internaltranscriptspacer2 (ITS-2)segmentofgenus FasciolosisribosomalDNA．Thesensitivity and specificity ofthedeveloped method wereexaminedusing the plasmid containing the targeted ITS一2 fragmentas template．The primers and probe were specific enoughtodistinguishFasciolosisfrom clonorchissinensis，fasciolopsisandparagonimuswestermani．The dynamic range ofthe developed method wasbetween 1xlO。and 1xlO copies，meaning thatthe target DNA could bereliably detected and quantified forplasmid template DNA ataconcentration aslow as one copy．Two fasciola oocystscould be detectedby thisrealtimePCR method．In conclusion，the real timePCR method israpid，sensitiveandspecificofrFasciolosis，and canbeusedto inspectFasciolosisin the farm and ofrquarantineuse． KeyWords：Fasciolosis；Real—timePCR；Sensitiviyt；Specificiyt；Detection 1 前言 寄生虫病。该病在世界各地均有