Nsp9基因实时荧光定量PCR检测方法的建立及其在感染细胞过程中表达量的变化

doi:10.16431/j.cnki.1671-7236.2016.10.004

《中国畜牧兽医》 ›› 2016, Vol. 43 ›› Issue (10): 2534-2540.doi: 10.16431/j.cnki.1671-7236.2016.10.004

Nsp9基因实时荧光定量PCR检测方法的建立及其在感染细胞过程中表达量的变化

赵孟孟^1,2, 冯松林¹, 王文佳³, 邢星⁴, 冯嘉萍¹, 张桂红¹

1. 华南农业大学兽医学院, 国家生猪种业工程技术研究中心, 广东省动物源性人兽共患病预防与控制重点实验室, 广州 510642;
2. 河南农业大学牧医工程学院, 郑州 450002;
3. 河南牧业经济学院制药工程学院, 郑州 450046;
4. 广西钦州保税港区出入境检验检疫局, 钦州 535008

收稿日期:2016-02-19 出版日期:2016-10-20 发布日期:2016-10-28
通讯作者: 张桂红 E-mail:guihongzh@scau.edu.cn
作者简介:赵孟孟(1986-),男,河南汝州人,博士,研究方向:猪繁殖与呼吸综合征病毒的致病机理,E-mail:zhaomengmeng502@163.com
基金资助:
国家自然科学基金猪繁殖与呼吸综合征病毒非结构蛋白Nsp9功能研究项目（31272564）；公益性行业（农业）科研专项经费（201203039）；国家生猪现代农业产业技术体系（CARS-36）项目；国家重点研发计划课题（2016YFD0500707）

Establishment of the Quantitative Real-time PCR Method for PRRSV Nsp9 Gene Rapid Detection and Its Expression in PRRSV Infected Cells

ZHAO Meng-meng^1,2, FENG Song-lin¹, WANG Wen-jia³, XING Xing⁴, FENG Jia-ping¹, ZHANG Gui-hong¹

1. Key Laboratory of Zoonosis Prevention and Control of Guangdong Province, National Engineering Research Center for Breeding Swine Industry, College of Veterinary, South China Agricultural University, Guangzhou 510642, China;
2. College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou 450002, China;
3. School of Pharmaceutical Engineering, Henan University of Animal Husbandry and Economy, Zhengzhou 450046, China;
4. Guangxi Qinzhou Free Trade Port Area Entry-exit Inspection and Quarantine Bureau, Qinzhou 535008, China

Received:2016-02-19 Online:2016-10-20 Published:2016-10-28

摘要/Abstract

摘要：

试验旨在建立猪繁殖与呼吸综合征病毒（porcine reproductive and respiratory syndrome virus，PRRSV）Nsp9基因的实时荧光定量PCR检测方法，并探究其在感染细胞过程中表达量的变化。根据PPRSVNsp9基因序列设计引物，建立基于SYBR Green Ⅰ检测模式的实时荧光定量PCR。结果显示，PRRSV Nsp9基因在1×10⁴~1×10⁸拷贝/μL范围内有很好的线性关系，扩增产物的熔解曲线只有1个单特异峰，无引物二聚体。该方法与猪戊肝病毒（HEV）、猪流感病毒（SIV）、伪狂犬病病毒（PRV）基因组均无交叉反应，可重复性好，组内变异系数小，可用于临床PRRSV检测。在病毒感染细胞过程中，Nsp9基因表达量逐步升高，36 h达到最高。本研究为探究病毒复制规律与临床疫苗生产奠定了理论基础。

关键词: 猪繁殖与呼吸综合征病毒; SYBR Green Ⅰ; 实时荧光定量PCR; Nsp9基因

Abstract:

The study was conducted to establish a quantitative Real-time PCR method for PRRSV Nsp9 gene rapid detection and study its expression in PRRSV infected cells.A pair of specific primers targeted to Nsp9 of PRRSV was designed and a Real-time PCR method based on SYBR Green Ⅰ fluorescent was developed for the quantization of PRRSV.The melting curve analysis using SYBR Green Ⅰ dye showed one specific peak,and no primer dimers peak was observed.No amplification was detected from HEV,SIV and PRV samples by this method.There was good reproducibility and low variation coefficient.The quantitative Real-time PCR method developed in this study would be useful for rapid laboratory diagnosis and epidemiology investigation of PRRSV.During the process of virus infecting cells,the expression level of Nsp9 increased gradually,and it got the highest at 36 h.This study laid the theoretical basis to explore the law of virus replication and clinical vaccine production.

Key words: PRRSV; SYBR Green Ⅰ; quantitative Real-time PCR; Nsp9 gene

中图分类号:

S852.65

赵孟孟, 冯松林, 王文佳, 邢星, 冯嘉萍, 张桂红. Nsp9基因实时荧光定量PCR检测方法的建立及其在感染细胞过程中表达量的变化[J]. 《中国畜牧兽医》, 2016, 43(10): 2534-2540.

ZHAO Meng-meng, FENG Song-lin, WANG Wen-jia, XING Xing, FENG Jia-ping, ZHANG Gui-hong. Establishment of the Quantitative Real-time PCR Method for PRRSV Nsp9 Gene Rapid Detection and Its Expression in PRRSV Infected Cells[J]. , 2016, 43(10): 2534-2540.

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Nsp9基因实时荧光定量PCR检测方法的建立及其在感染细胞过程中表达量的变化

Establishment of the Quantitative Real-time PCR Method for PRRSV Nsp9 Gene Rapid Detection and Its Expression in PRRSV Infected Cells

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