大熊猫轮状病毒PCR检测方法的建立及应用

苏小艳; 李林; 燕霞; 张东升; 侯蓉; 刘颂蕊

doi:10.16829/j.slxb.150454

兽类学报 >

2021 , Vol. 41 >Issue 3: 254 - 260

DOI: https://doi.org/10.16829/j.slxb.150454

动物疫病专题

大熊猫轮状病毒PCR检测方法的建立及应用

苏小艳 ,
李林 ,
燕霞 ,
张东升 ,
侯蓉 ,
刘颂蕊

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1 成都大熊猫繁育研究基地, 成都 610081;
2 四川省濒危野生动物保护生物学重点实验室, 成都 610081;
3 四川省大熊猫科学研究院, 成都 610081

苏小艳(1985-),助理研究员,主要从事大熊猫等野生动物疾病防控工作.

收稿日期: 2020-07-03

网络出版日期: 2021-05-27

基金资助

四川省科技计划项目（面上）（2018JY0363）；成都大熊猫繁育研究基地自立课题项目（2020CPB-B22）

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Establishment and application of giant panda rotavirus PCR detection method

SU Xiaoyan ,
LI Lin ,
YAN Xia ,
ZHANG Dongsheng ,
HOU Rong ,
LIU Songrui

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1 Chengdu Research Base of Giant Panda Breeding, Chengdu 610081, China;
2 Sichuan Key Laboratory of Conservation Biology for Endangered Wildlife, Chengdu 610081, China;
3 Sichuan Academy of Giant Panda, Chengdu 610081, China

Received date: 2020-07-03

Online published: 2021-05-27

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摘要

轮状病毒是威胁大熊猫健康的主要病原微生物之一。为了对大熊猫轮状病毒进行快速、方便且准确地检测，研发适合于基层饲养单位和保护区的检测方法是非常有必要的。本研究通过合成大熊猫轮状病毒Vp7基因序列，构建了PUC-VP7的重组质粒，并将其作为阳性对照对大熊猫轮状病毒样本进行PCR检测和分析。结果表明，在进行PCR扩增分析时，该质粒和病毒cDNA二者均在340 bp处出现了特异性条带。此外，对收集到的45份大熊猫粪便样本进行轮状病毒抗原检测时，其中2份样品在340 bp处出现条带，该基因片段与大熊猫轮状病毒CH-1株的相似性为99.89%。本研究构建的PUC-VP7质粒不但可以作为大熊猫轮状病毒PCR检测中的阳性质控品，而且还能有效地促进该PCR病毒检测技术在基层饲养单位和保护区的推广和应用。

关键词： 大熊猫轮状病毒; PCR检测; Vp7基因; 质粒构建

本文引用格式

苏小艳 , 李林 , 燕霞 , 张东升 , 侯蓉 , 刘颂蕊 . 大熊猫轮状病毒PCR检测方法的建立及应用[J]. 兽类学报, 2021 , 41(3) : 254 -260 . DOI: 10.16829/j.slxb.150454

Abstract

Rotavirus is one of the main pathogenic microorganisms threatening the survival of giant pandas. In order to detect panda rotavirus antigens quickly, conveniently and accurately, it is necessary to develop a detection method suitable for ex-situ breeding centers and protection areas. In this study, the panda rotavirus Vp7 gene sequence was synthesized to construct the PUC-VP7 recombinant plasmid, and it was used as a positive control for PCR detection and analysis of panda rotavirus samples. The results showed that during the PCR amplification analysis, both the plasmid and the viral cDNA showed a specific band at 340 bp. In addition, when performing rotavirus antigen detection on forty-five panda rotavirus fecal samples, two samples showed a band at 340 bp, and the gene fragment was 99.89% homologous to the panda rotavirus CH-1 strain. The PUC-VP7 plasmid constructed in this study can not only be used as a positive quality control material in the PCR detection of giant panda rotavirus, but can also effectively improve the promotion and application of the PCR virus detection technology in ex-situ breeding centers and protected areas.

Key words： Giant panda rotavirus; PCR test; Vp7 gene; Plasmid construction

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