蜱携带牛丙型肝炎病毒新亚型巢式PCR检测方法的建立  

The Development of Nested PCR Assay for New Subtype of Bovine Hepacivirus in Ticks

作　　者：郭銮英 王妮娜 李杭远 纪雨霏 马骏 裴明超 邵建伟 刘全 GUO Luanying;WANG Nina;LI Hangyuan;JI Yufei;MA Jun;PEI Mingchao;SHAO Jianwei;LIU Quan(College of Life Sciences and Engineering, Foshan University, Foshan 528000, China)

机构地区：[1]佛山科学技术学院生命科学与工程学院,佛山528000

出　　处：《畜牧兽医学报》2022年第3期972-977,共6页ACTA VETERINARIA ET ZOOTECHNICA SINICA

基　　金：广东省珠江人才计划项目(2019CX01 N111);佛山科学技术学院2020年度学生学术基金(xsjj202009kja01)。

摘　　要：旨在提高新发现的牛丙型肝炎病毒毒株——广东湛江毒株(bovine hepacivirus-GDZJ,BovHepV-GDZJ)的检测及诊断能力,初步了解广东地区蜱携带BovHepV-GDZJ的情况,本研究结合宏转录组学、高通量测序及PCR技术,针对高通量测序结果中丰度高的基因区域,设计该病毒的巢式PCR特异性引物。通过优化巢式PCR方法的最佳退火温度,建立检测BovHepV-GDZJ的特异性巢式PCR检测方法。结果显示:本方法对该病毒标准质粒最低检测值为6.8 copies·μL^(-1);对沙粒病毒等蜱携带的常见病原均无反应;应用此方法对115份广东湛江的蜱样品进行了检测,测序结果显示,该批蜱携带BovHepV阳性率为46.08%(53/115)。本研究首次在蜱中检测到BovHepV,揭示了BovHepV可能的传播途径,并建立了一套针对BovHepV-GDZJ株的特异性巢式PCR检测方法,为广东地区检测和预防BovHepV提供技术帮助,也为控制BovHepV的流行提供参考。A detection method is urgently needed,which not only can improve the detection and diagnostic ability for the newly discovered bovine hepacivirus strain GDZJ,BovHepV-GDZJ,but also contribute to the preliminary understanding of tick carrying BovHepV in Guangdong province.In this study,the specific primers of Nested PCR for BovHepV-GDZJ strain were designed using the gene regions with high abundance in high-throughput sequencing data as templates through macrotranscriptomics,high-throughput sequencing,and PCR.The results showed that the minimum detection value of the method for the standard plasmid containing the gene fragment of BovHepV-GDZJ strain was 6.8 copies·μL^(-1),and it had no response to common pathogens carried by ticks such as arenavirus.Moreover,the positive rate was 46.08%(53/115)in the 115 tick samples collected in Zhanjiang,Guangdong.In this study,BovHepV was first detected from ticks,which revealing the possible transmission route of BovHepV.What’s more,a nested PCR method for the detection of BovHepV-GDZJ strain was established to provide technical help for the detection and prevention of BovHepV in Guangdong.It also provides some reference value for the control of the HNV epidemic.

关 键 词：牛丙型肝炎病毒 巢式PCR 蜱 高通量测序 

分 类 号：S852.653[农业科学—基础兽医学]