基于恒温隔绝式荧光RT-PCR技术现场检测牛纽布病毒方法的建立及应用

doi:10.11843/j.issn.0366-6964.2022.04.032

摘要/Abstract

摘要： 纽布病毒(nebovirus,NeV)是引起犊牛腹泻的新发病原，本试验旨在建立基于恒温隔绝式RT-PCR (insulated isothermal RT-PCR,iiRT-PCR)技术现场检测NeV的方法。根据NeV的RdRp基因的保守区域，设计并合成引物和探针，优化检测体系和检测试剂预混方法，建立检测NeV的iiRT-PCR方法。结果显示，成功建立了检测NeV的iiRT-PCR方法，该方法只特异性检出NeV，对牛冠状病毒、牛诺如病毒、A群牛轮状病毒、牛病毒性腹泻病毒、牛凸隆病毒、牛大肠杆菌、牛沙门菌、牛瑞氏隐孢子虫和牛艾美尔球虫等无关病原不检出，批间变异系数为3.07%~3.12%，批内变异系数为2.45%~3.01%，检测下限为5.38 copies·μL^-1。应用此方法对2020—2021年采集自红原县、若尔盖县、凉山彝族自治州西昌市和阆中市的101份犊牛腹泻样本进行检测，NeV的检出率为64.36%。本研究成功建立了检测NeV的iiRT-PCR方法，具有特异性强、稳定性好和灵敏度高的特点，检测试剂预混后配合使用PetNAD核酸萃取试剂盒，可实现NeV的现场检测，从核酸提取到报告检测结果仅需1 h，操作方便，为NeV的快速诊断提供了有力的工具。

关键词: 纽布病毒, 牛, RdRp基因, 恒温隔绝PCR, 现场检测

Abstract: Nebovirus (NeV) is an emerge diarrhea-causing virus in calves, the aim of this study is to establish an insulated isothermal RT-PCR(iiRT-PCR) for detecting NeV on field. Based on the RdRp sequences of NeV in GenBank database, a pair of primers and a fluorescent TaqMan probe were designed and synthesized. After optimizing the react system and condition, the iiRT-PCR method for detection of NeV was established. The iiRT-PCR assay could amplify specific fragment of NeV, without amplification of irrelevant pathogens, including bovine coronavirus, bovine norovirus, bovine rotavirus, bovine viral diarrhea virus, bovine torovirus, bovine Cryptosporidium parvum, bovine Eimeria. The intra- and inter-coefficients of variation were 3.07%-3.12% and 2.45%-3.01%, respectively, and the detection limit of viral nucleic acid of the assay was 5.38 copies·μL^-1. One hundred and one calf diarrhea samples, collected from Hongyuan County, Ruoergai Prefecture, Xichang City, Liangshan Yi Autonomous Prefecture and Langzhong City in Sichuan Province during 2020-2021, were used to detect NeV, and 64.36% samples were detected as NeV positive. The study established an iiRT-PCR method for NeV detection with good specificity and reproducible as well as high sensitivity. Moreover, combined with the premixed detection reagent and PetNAD nucleic acid extraction kit, this assay could be used to NeV detect on-site, and only 1 hour from nucleic acid extraction to result report,which contribute to the fast detection for NeV.

Key words: nebovirus, bovine, RdRp gene, iiRT-PCR, field test

中图分类号:

S852.659.6

崔雨晨, 岳华, 汤承. 基于恒温隔绝式荧光RT-PCR技术现场检测牛纽布病毒方法的建立及应用[J]. 畜牧兽医学报, 2022, 53(4): 1310-1316.

CUI Yuchen, YUE Hua, TANG Cheng. Development and Application of an Insulated Isothermal RT-PCR for On-site Detecting Nebovirus[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(4): 1310-1316.

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