猪嵴病毒RPA快速诊断方法的建立和应用  

Development and application of a recombinase polymerase amplification assay for rapid diagnosis of porcine kobuvirus

作　　者：刘占旭 刘新生[2] 方玉珍[2] 周鹏[2] 张巧玲 张丽萍 董昭良 张永光[2] 杜晓华[1] 王永录[2] LIU Zhan-xu;LIU Xin-sheng;FANG Yu-zhen;ZHOU Peng;ZHANG Qiao-ling;ZHANG Li-ping;DONG Zhao-liang;ZHANG Yong-guang;DU Xiao-hua;WANG Yong-lu(College of Life Science and Technology,Gansu Agricultural University,Lanzhou 730070,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]甘肃农业大学动物医学院,甘肃兰州730070 [2]中国农业科学院兰州兽医研究所家畜疫病病原生物学国家重点实验室,甘肃兰州730046

出　　处：《中国兽医科学》2018年第9期1073-1079,共7页Chinese Veterinary Science

基　　金：国家自然科学基金项目(31602095);国家生猪产业体系项目(CARS-36-068);“十三五”国家重点研发计划(2YFD0160501505);中国农业科学院兰州兽医研究所统筹项目(Y2016CG23)

摘　　要：根据GenBank中公布的猪嵴病毒（porcine kobuvirus，PKV）基因组序列，设计并合成特异性引物,构建阳性质粒标准品，以阳性标准品为模板建立猪嵴病毒的重组酶聚合酶扩增（RPA）快速检测方法，并对此方法的敏感性、特异性及重复性进行了评价。结果显示，以猪流行性腹泻病毒、猪传染性胃肠炎病毒、猪德尔塔冠状病毒、口蹄疫病毒、猪繁殖与呼吸综合征病毒为模板时均未检测到特异性扩增条带，表明该方法具有良好的特异性；用3．36×10^7、3．36×10^6和3．36×10^5copies／μL三个不同浓度的质粒标准品进行重复试验，均能扩增出特异性条带，表明该方法具有良好的重复性。10倍梯度稀释质粒标准品．此方法检测的最低限度为3．36×10^2copies／pL的质粒DNA，表明此方法具有良好的敏感性。利用该方法对208份临床猪粪便样品进行了检测，结果显示，猪嵴病毒阳性率为73．1％，明显高于常规RT-PCR的46．6％，两种方法的符合率为90．2％。上述结果表明，本研究建立的RPA方法可应用于猪嵴病毒的临床诊断及快速检测。According to porcine kobuvirus reference sequences published on GenBank,the specific primers were designed and synthesized,and the positive plasmid-standard was constructed. The RPA rapid detection method for porcine kobuvirus was established using the positive standard as a template,and the sensitivity,specificity and repeatability of this method were evaluated. In result,no specific amplification bands were detected using porcine epidemic diarrhea virus,porcine transmissible gastroenteritis virus,porcine delta coronaVirus,porcine foot-and-mouth disease virus and porcine reproductive and respiratory syndrome virus. Using different concentrations of plasmid standards（3.36×10^7,3.36×10^6 and 3.36×10^5copies/μL）as templates,the good specificity and repeatability were shown in this method. The sensitivity of the assays was evaluated using 10-fold serial dilutions of plasmid standard and the results showed that the detection limits of this method was 3.36×10^2 copies/μL of plasmid DNA. It indicated that this method had good sensitivity. Besides,208 samples of pig stool samples were detected using this method and the results showed that the positive rate of porcine kobuvirus was 73.1% and significantly higher than that detected by the conventional RT-PCR （46.6%）.The coincidence rate between this method and RT-PCR was 90.2%.According to the above results,the developed RPA method can be applied to clinical diagnosis and rapid detection of porcine kobuvirus.

关 键 词：重组酶聚合酶扩增 猪嵴病毒 快速检测 

分 类 号：S852.651[农业科学—基础兽医学]