中国兽医科学2018，Vol.48Issue(9)：1079-1079,1.DOI:10.16656/j.issn.1673-4696.2018.0135
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
摘要
Abstract
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× 107,3. 36× 106 and 3. 36× 105 copies/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 × 102 copies/μ L 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Key words
recombinase polymerase amplification (RPA)/porcine kobuvirus/rapid detection分类
农业科技引用本文复制引用
刘占旭,王永录,刘新生,方玉珍,周鹏,张巧玲,张丽萍,董昭良,张永光,杜晓华..猪嵴病毒RPA快速诊断方法的建立和应用[J].中国兽医科学,2018,48(9):1079-1079,1.基金项目
国家自然科学基金项目(31602095) （31602095）
国家生猪产业体系项目(CARS-36-068) （CARS-36-068）
"十三五"国家重点研发计划(2YFD0160501505) （2YFD0160501505）
中国农业科学院兰州兽医研究所统筹项目(Y2016CG23) （Y2016CG23）
