牛副流感病毒3型3种基因型多重RT-PCR检测方法的建立

doi:10.16431/j.cnki.1671-7236.2018.01.004

《中国畜牧兽医》 ›› 2018, Vol. 45 ›› Issue (1): 32-38.doi: 10.16431/j.cnki.1671-7236.2018.01.004

牛副流感病毒3型3种基因型多重RT-PCR检测方法的建立

杨帆^1,2, 石顺利³, 徐娜^1,2, 雷宇^1,2, 常塔娜^1,2, 李平安^1,2, 关平原^1,2

1. 内蒙古农业大学兽医学院, 呼和浩特 010018;
2. 农业部动物疾病临床诊疗技术重点实验室, 呼和浩特 010018;
3. 内蒙古自治区通辽市家畜繁育指导站, 通辽市 028000

收稿日期:2017-07-10 出版日期:2018-01-20 发布日期:2018-01-20
通讯作者: 关平原 E-mail:gpynm@sina.com
作者简介:杨帆(1992-),女,内蒙古乌兰察布人,硕士,研究方向:动物传染病的诊断与防控,E-mail:735519114@qq.com
基金资助:
内蒙古自治区自然科学基金项目"内蒙古地区牛副流感的分子流行病学研究"（2016MS347）

Establishment of a Multiplex RT-PCR Detection Method for Three Genotypes of Bovine Parainfluenza Virus Type 3

YANG Fan^1,2, SHI Shunli³, XU Na^1,2, LEI Yu^1,2, CHANG Tana^1,2, LI Pingan^1,2, GUAN Pingyuan^1,2

1. College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010018, China;
2. Key Laboratory of Clinical Diagnosis and Treatment Technology in Animal Disease, Ministry of Agriculture, Hohhot 010018, China;
3. Livestock Breeding Station of Tongliao City in Inner Mongolia, Tongliao 028000, China

Received:2017-07-10 Online:2018-01-20 Published:2018-01-20

摘要/Abstract

摘要：

为建立检测牛副流感病毒3型（bovine parainfluenza virus type 3，BPIV3）3种基因型的多重RT-PCR方法，根据GenBank上发表的BPIV3 3种基因型病毒株的HN基因序列设计特异性引物，优化反应体系建立多重RT-PCR方法。结果显示，方法可同时扩增出BPIV3 A型150 bp、B型253 bp和C型342 bp的特异性片段，与牛传染性鼻气管炎病毒（IBRV）、牛呼吸道合胞体病毒（BRSV）、牛病毒性腹泻病毒（BVDV）、小反刍兽疫病毒（PPRV）、牛支原体、牛布鲁氏菌、羊布鲁氏菌、牛源多杀性巴氏杆菌A型和B型均无交叉反应，A、B、C基因型BPIV3最低阳性质粒检测量分别为0.89×10⁴、0.92×10⁴和1.53×10⁴拷贝/μL。本试验建立的多重RT-PCR检测方法操作方便、特异性强，应用于临床样本的检测，可快速检测BPIV3 3种基因型。

关键词: 牛副流感病毒3型(BPIV3); 基因型; 多重RT-PCR; 检测

Abstract:

In order to develop a multiplex RT-PCR method for detecting three genotypes of bovine parainfluenza virus type 3 (BPIV3), according to the hemagglutinin-neuraminidase protein genomic sequences of three BPIV3 genotypes obtained from GenBank, three pairs of specific PCR primers were designed. A multiplex RT-PCR detecting three genotypes of BPIV3 was established and optimized. The results showed no cross-reactivity with IBRV, BRSV, BVDV, M.bovis, PPRV, B.melitensis, B.abortus, bovine P.multocida serotype A and bovine P.multocida serotype B. In this study, the amplification produced a series of specific fragments with lengths of 150 bp (BPIV3a), 253 bp (BPIV3b) and 342 bp(BPIV3c), respectively. The limit detection of three recombinant plasmids were 0.89×10⁴, 0.92×10⁴ and 1.53×10⁴ copies/μL. This multiplex RT-PCR method was high specificity and simplicity of operator. It was applied to detect clinical samples and could rapidly detect three genotypes of BPIV3.

Key words: bovine parainfluenza virus type 3 (BPIV3); genotypes; multiplex RT-PCR; detection

中图分类号:

S858.23

杨帆, 石顺利, 徐娜, 雷宇, 常塔娜, 李平安, 关平原. 牛副流感病毒3型3种基因型多重RT-PCR检测方法的建立[J]. 《中国畜牧兽医》, 2018, 45(1): 32-38.

YANG Fan, SHI Shunli, XU Na, LEI Yu, CHANG Tana, LI Pingan, GUAN Pingyuan. Establishment of a Multiplex RT-PCR Detection Method for Three Genotypes of Bovine Parainfluenza Virus Type 3[J]. , 2018, 45(1): 32-38.

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牛副流感病毒3型3种基因型多重RT-PCR检测方法的建立

Establishment of a Multiplex RT-PCR Detection Method for Three Genotypes of Bovine Parainfluenza Virus Type 3

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