畜牧兽医学报2017，Vol.48Issue(6)：1092-1098,7.DOI:10.11843/j.issn.0366-6964.2017.06.014
基于恒温隔绝式荧光PCR技术现场检测牛轮状病毒方法的建立及应用
Development and Application of an Insulated Isothermal PCR (iiPCR)for On-site Detecting Bovine Rotavirus
摘要
Abstract
Rotavirus (RV) is an important cause of calves diarrhea.The objective of this study was to establish an insulated isothermal PCR (iiPCR) assay for on-site detecting bovine rotavirus (BRV).A pair of primers and a fluorescent TaqMan probe were designed according to BRV and Yak RV VP6 gene sequences.After optimizing the reaction system and nucleic acid extraction by a commercial PetNAD nucleic acid extraction kit,the iiPCR assay for detecting BRV and yak RV was successfully established.The results showed that the assay could amplify specific fragment of BRV and yak RV,with no amplification of other unrelated pathogens,including bovine coronavirus,bovine astrovirus,bovine viral diarrhea virus,Escherichia coli,Salmonella,Eimeria and Cryptosporidium.The detection limit of viral nucleic acid of the assay was 96.6 copies · μL-1 with good reproducibility.Thirty clinical diarrhea samples of calves and 30 clinical diarrhea samples of yaks were used to evaluate the coincidence of this assay with two reported assays,and the positive rate of BRV and yak RV samples was 33.33％ and 73.33％ respectively by this assay,which has 100％ coincident detection rate with the reported assays for detecting BRV or Yak RV,respectively.Further,98.86％ positive rate of yak RV was detected by the assay developed in this study in 88 yak diarrhea samples from 14 pastures in Northwest Sichuan plateau in 2016.The established iiPCR assay in this study was specific,sensitive and reproducible,and it can be used for detecting not only BRV but also yak RV.It takes only 1 hour from nucleic acid extraction to report test result with easy operation,which makes it a valuable tool for rapid on-site diagnostics of BRV.关键词
牛/牦牛/轮状病毒/VP6基因/恒温隔绝PCR/现场检测Key words
calf/yak/rotavirus/VP6 gene/iiPCR/on-site detection分类
农业科技引用本文复制引用
李凡,岳华,邱莞思,张斌,黄建德,汤承..基于恒温隔绝式荧光PCR技术现场检测牛轮状病毒方法的建立及应用[J].畜牧兽医学报,2017,48(6):1092-1098,7.基金项目
十三五国家重点研发计划(2016YFD0500907) （2016YFD0500907）
国家民委科研项目(14XNZ025) （14XNZ025）
