犬源牛犬细小病毒和犬圆环病毒二联PCR检测方法的建立及应用

doi:10.11843/j.issn.0366-6964.2022.03.031

畜牧兽医学报 ›› 2022, Vol. 53 ›› Issue (3): 978-982.doi: 10.11843/j.issn.0366-6964.2022.03.031

犬源牛犬细小病毒和犬圆环病毒二联PCR检测方法的建立及应用

于永乐¹, 姚延珠², 韩先杰¹, 张传美¹, 秦志华¹, 杨瑞梅¹, 张洪亮¹, 刘维全^3*, 单虎^1*

1. 青岛农业大学动物医学院山东省预防兽医学重点实验室, 青岛 266109;
2. 青岛农业大学植物医学学院, 青岛 266109;
3. 中国农业大学生物学院农业生物技术国家重点实验室, 北京 100193

收稿日期:2021-05-25 出版日期:2022-03-23 发布日期:2022-03-31
通讯作者: 单虎,主要从事动物传染病学研究,E-mail:shanhu67@163.com;刘维全,主要从事动物病毒学研究,E-mail:weiquan8@126.com
作者简介:于永乐(1988-),男,博士,主要从事动物病毒学研究,E-mail:1511832711@qq.com;姚延珠(1987-),女,博士,主要从事细胞生物学研究,E-mail:yzyao2011@163.com
基金资助:
在青高校服务青岛重点学科(兽医学)(025/1119002);"十三五"国家重点研发计划资助项目(2016YFD0501001;2016YFD0501004);青岛农业大学高层次人才科研基金(1120015/663)

Establishment of Duplex PCR Detective Techniques for Canine Bocavirus and Canine Circovirus

YU Yongle¹, YAO Yanzhu², HAN Xianjie¹, ZHANG Chuanmei¹, QIN Zhihua¹, YANG Ruimei¹, ZHANG Hongliang¹, LIU Weiquan^3*, SHAN Hu^1*

1. College of Veterinary Medicine, Qingdao Agricultural University, Qingdao 266109, China;
2. College of Plant Health and Medicine, Qingdao Agricultural University, Qingdao 266109, China;
3. State Key Laboratory of Agrobiotechnology, Department of Biochemistry and Molecular Biology, College of Biological Sciences, China Agricultural University, Beijing 100193, China

Received:2021-05-25 Online:2022-03-23 Published:2022-03-31

摘要/Abstract

摘要： 本研究旨在建立可同时检测犬源牛犬细小病毒(CBoV)和犬圆环病毒(CCV)的二联PCR检测方法,并对两种病毒病当前的流行情况进行监测和调查。分别将已发表的CBoV和CCV基因组序列进行同源性比对,选择高同源区段,应用Primer Primier 5计算机软件设计并合成了2对特异性扩增引物,目的片段大小分别为170 bp (CBoV)和239 bp (CCV)。分别提取CBoV和CCV重组质粒作为模板,进行二联PCR扩增试验。结果表明,本研究成功建立了可同时检测CBoV和CCV的二联PCR检测方法。并对其重复性、特异性和灵敏度进行验证,二联PCR最低检测限度为3.0 pg总DNA,而对犬细小病毒2型、犬瘟热病毒和犬副流感病毒的PCR检测结果均为阴性。对送检的30份病料进行检测,其中,4份为CBoV阳性,3份为CCV阳性,未检测到混合感染。综上所述,本研究所建立的CBoV和CCV二联PCR诊断方法特异性高,敏感性好,可用于临床犬腹泻病的流行病学调查。

关键词: 犬源牛犬细小病毒, 犬圆环病毒, 二联PCR

Abstract: This study was conducted to develop the duplex PCR assay for detection simultaneously and discrimination of canine bocavirus (CBoV) and canine circovirus (CCV). The published gene sequences of CBoV and CCV in GenBank were analyzed and compared with each other, respectively. Two pairs of specific primers were designed with the Primer Primier 5 software for PCR amplification. The results showed that two specific fragments of the two viruses were simultaneously amplified and the length of amplified fragments were as follows:170 bp for CBoV and 239 bp for CCV. The sensitivity and specificity tests showed that the assay could detect at least 3.0 pg of target DNA, and there was no cross-reaction with those clinically relevant pathogens, ie. canine parvovirus type 2, canine distemper virus and canine parainfluenza virus. The detection results for 30 clinical samples with the duplex PCR showed that 4 samples were positive for CBoV and 3 for CCV. These results indicated that the method could provide technical support for the detection of CBoV and CCV, and epidemiological investigation of canine viral diarrhea.

Key words: canine bocavirus, canine circovirus, duplex PCR

中图分类号:

S852.655

于永乐, 姚延珠, 韩先杰, 张传美, 秦志华, 杨瑞梅, 张洪亮, 刘维全, 单虎. 犬源牛犬细小病毒和犬圆环病毒二联PCR检测方法的建立及应用[J]. 畜牧兽医学报, 2022, 53(3): 978-982.

YU Yongle, YAO Yanzhu, HAN Xianjie, ZHANG Chuanmei, QIN Zhihua, YANG Ruimei, ZHANG Hongliang, LIU Weiquan, SHAN Hu. Establishment of Duplex PCR Detective Techniques for Canine Bocavirus and Canine Circovirus[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(3): 978-982.

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犬源牛犬细小病毒和犬圆环病毒二联PCR检测方法的建立及应用

Establishment of Duplex PCR Detective Techniques for Canine Bocavirus and Canine Circovirus

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