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

doi:10.11843/j.issn.0366-6964.2017.06.014

摘要/Abstract

摘要：

轮状病毒（rotavirus，RV）是犊牛腹泻的重要病因，笔者旨在建立基于恒温隔绝PCR（insulated isothermal PCR，iiPCR）技术现场检测牛轮状病毒（bovine rotavirus，BRV）的方法。根据BRV和牦牛RV VP6基因序列，设计合成引物和探针，通过优化反应体系，配合商品化PetNAD核酸萃取试剂盒，建立了检测BRV和牦牛RV的iiPCR方法。结果显示该方法只能检出BRV和牦牛RV，不能检出牛冠状病毒、牛星状病毒、牛病毒性腹泻-黏膜病病毒、牛大肠杆菌、牛沙门菌、牛艾美尔球虫、牛瑞氏隐孢子虫等无关病原；检测下限为96.6 copies·μL^-1，重复性好；对30 份牛腹泻样本和 30 份牦牛腹泻样本的检测结果表明，本方法对BRV的检出率是33.33%，对牦牛RV的检出率为73.33%，与文献报道的检测BRV和牦牛RV的方法的符合率分别为100%；对2016年四川阿坝藏羌自治州14个牧场的88份犊牦牛腹泻样本RV的检测结果显示RV核酸阳性检出率为98.86%。本研究建立的BRV iiPCR方法特异性好、灵敏度高、重复性好，既可用于BRV检测，又可用于牦牛RV的检测；从核酸提取到报告检测结果仅需1 h，操作方便，可现场使用，为BRV的现场快速诊断提供有力的工具。

关键词: 牛, 牦牛, 轮状病毒, VP6基因, 恒温隔绝PCR, 现场检测

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.

Key words: calf, yak, rotavirus, VP6 gene, iiPCR, on-site detection

中图分类号:

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.

LI Fan, YUE Hua, QIU Wan-si, ZHANG Bin, HUANG Jian-de, TANG Cheng. Development and Application of an Insulated Isothermal PCR (iiPCR) for On-site Detecting Bovine Rotavirus[J]. ACTA VETERINARIA ET ZOOTECHNICA SINICA, 2017, 48(6): 1092-1098.

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