摘要
目的建立一种多重聚合酶链式反应(multiplex polymerase chain reaction,mPCR)方法,用于快速鉴别猪囊尾蚴等4种猪的绦虫蚴。方法比较猪带绦虫(Taenia solium)、亚洲带绦虫(Taenia asiatica)、细粒棘球绦虫(Echinococcus granulosus)和泡状带绦虫(Taenia hydatigena)的线粒体基因组,选取tRNA-Val、tRNA-Ala、tRNA-Asp和nad1基因作为分子检测靶标,设计4条种特异性上游引物(TaF、EgF、ThF、和TsF)和1条下游通用引物(TR),通过优化反应条件和体系,建立了在一个PCR体系中即可完成对4种猪绦虫蚴的快速鉴别的mPCR方法。结果4种猪绦虫蚴的特异性PCR引物等比例混合后,在退火温度为50℃的条件下,该mPCR方法可同时扩增出C.cellulosae、T.asiatica、E.granulosus和C.tenuicollis的特异性基因片段,其大小分别为478 bp、710 bp、619 bp和542 bp。PCR产物测序后经BLAST比对,与同种虫体的基因序列相似性在99%以上。结论本研究建立的mPCR方法具有很高的种间特异性,并且可通过扩增片段的大小对4种猪绦虫蚴进行快速、准确的鉴别。
A multiplex polymerase chain reaction(mPCR)method was established for rapid identification of four porcine metacestodes.Four specific upstream primers(TaF,EgF,ThF and TsF)and one universal downstream primer(TR)were designed on the basis of the tRNA-Val,tRNA-Ala,tRNA-Asp and nad1 genes by comparing the mitochondrial genome of Taenia solium(T.solium)Taenia asiatica(T.asiatica)Echinococcus granulosus(E.granulosus)and Taenia hydatigena(T.hydatigena).The mPCR method was established in one PCR system by optimizing PCR conditions.Using the annealing temperature of 50℃and equal-proportion mixing of four metacestode-specific upstream primers conditions,the specific gene fragments of C.cellulosae,T.asiatica,E.granulosus and C.tenuicollins were amplified simultaneously.Their fragment sizes were 478 bp,710 bp,619 bp and 542 bp,respectively.Furthermore,the similarity of PCR product sequences after sequencing and aligning was>99%in the same species.The mPCR method established in this study has a high interspecies specificity and can quickly and accurately identify four species of swine tapeworm larvae by the size of their amplified fragments.
作者
陈国梁
郭爱民
张少华
梁盼红
李艳萍
王立群
刘婷丽
白雪
骆学农
CHEN Guo-liang;GUO Ai-min;ZHANG Shao-hua;LIANG Pan-hong;LI Yan-ping;WANG Li-qun;LIU Ting-li;BAI Xue;LUO Xue-nong(Key Laboratory of Veterinary Parasitology of Gansu Province/State Key Laboratory of Veterinary Etiological Biology/Lanzhou Veterinary Research Institute,Chinese Academy of Agricultural Sciences,Lanzhou 730046,China;Institute of Zoonosis,Jilin University,Changchun 130062,China)
出处
《中国人兽共患病学报》
CAS
CSCD
北大核心
2021年第6期496-501,共6页
Chinese Journal of Zoonoses
基金
国家重点研发计划项目(No.2017FYD0501303,No.2017YFC1601206)。
关键词
猪
4种绦虫蚴
多重PCR
鉴别诊断
porcine
four metacestodes
multiplex PCR
differential diagnosis
