简并引物q-PCR法检测新疆喀什地区羊肝组织中细粒棘球绦虫  

Detection of Echinococcus granulosus in sheep liver from Kashi region of Xinjiang by degenerate primer q-PCR method

作　　者：王会品[1] 李欣 孙世珺[1] WANG Hui-pin;LI Xin;SUN Shi-jun(Molecular Diagnosis Center,Zhongshan People′s Hospital,Zhongshan 528400,Guangdong,China;不详)

机构地区：[1]中山市人民医院分子诊断中心,广东中山528400 [2]中山市人民医院耳鼻咽喉科,广东中山528400

出　　处：《广东医学》2022年第1期46-50,共5页Guangdong Medical Journal

基　　金：中山市社会公益科技研究项目(2018B1083)。

摘　　要：目的建立检测新疆喀什地区羊肝组织中细粒棘球绦虫的分子生物学方法,提高包虫病的早期诊断水平。方法对羊肝组织内获取棘球蚴囊,将囊内容物离心沉淀提取DNA,以细粒棘球绦虫线粒体cytochrome b基因为靶基因设计引物进行PCR扩增,引物设计考虑简并碱基。结果建立的简并引物q-PCR法特异性好,与常见的宿主动物(羊、驴和犬)基因组无交叉扩增现象,与肝内的常见寄生虫(肝片吸虫和莫尼茨绦虫)基因组无交叉扩增。梯度稀释的基因组DNA:10 ng、1 ng、100 pg、10 pg进行qPCR实验均能得到标准的S型曲线。PCR体系R^(2)=0.984,效率:92.6%,符合qPCR的相关要求。结论线粒体基因组的cytochrome b基因分析是棘球蚴病诊断和分型的有力工具。喀什地区首次应用简并引物q-PCR法检测细粒棘球绦虫,灵敏度高,特异性好,为下一步对该地区进行包虫病分子流行病调查提供了技术支撑。Objective To establish a molecular biological method of detecting Echinococcus granulosus in sheep liver tissue in Kashgar,Xinjiang,and to improve the early diagnosis of hydatid disease.Methods Echinococcus granulosus capsules were obtained in sheep liver tissue,and the DNA was extracted.The cytochrome b gene was applied for PCR.Results The degenerate primer q-PCR method was established with good specificity,with no cross-amplification of host animals(sheep,donkey and dog),neither was with common parasites in the liver(Fasciola hepatica and Monitz tapeworm).The standard S type curves were obtained usign Grade-gradient diluted genome DNA:10 ng,1 ng,100 pg and 10 pg.In this PCR system,R^(2) was 0.98 with the efficiency of 92.6%.Conclusion The cytochrome b gene analysis of mitochondrial genome is a powerful tool for the diagnosis and typing of hydatid disease.The degenerate primer q-PCR method is used to detect the Echinococcus granulosus,with high sensitivity and good specificity,which provides technical support for the next hydatid molecular epidemic survey in the region.

关 键 词：细粒棘球蚴 CYTOCHROME B基因 基因型 分子流行病学 

分 类 号：R34[医药卫生—基础医学] R381