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大熊猫轮状病毒荧光定量RT-PCR检测方法的建立

陈珍容 廖红 郝中香 马磊 龚永平 王成东 陈欣 韩国全 兰景超 颜其贵   

  1. 四川农业大学动物医学院
  • 出版日期:2016-11-13 发布日期:2016-11-16
  • 通讯作者: 颜其贵 E-mail:yanqigui@126.com
  • 基金资助:
    国家科技支撑计划项目 (2012BAC01B06);973计划前期研究专项(2012CB722207);成都大熊猫繁育基金研究会项目CFP研2012-9 与CFP研2012-12

Establishment of fluorescent quantitative RT-PCR assay for detection of the giant panda rotavirus

CHEN Zhenrong, LIAO Hong, HAO Zhongxiang, MA Lei, GONG Yongping, WANG Chengdong, CHEN Xin, HAN Guoquan, LAN Jingchao, YAN Qigui   

  1. College of Veterinary Medicine, Sichuan Agricultural University
  • Online:2016-11-13 Published:2016-11-16
  • Contact: YAN Qigui E-mail:yanqigui@126.com

摘要: 为了能对大熊猫轮状病毒的早期感染和隐性感染大熊猫做出有效诊断,并为病毒定量分析提供技术支持。本研究根据 GenBank中登录的大熊猫轮状病毒(GPRV)VP4基因序列设计1对引物,建立一种快速准确的检测大熊猫轮状病毒的荧光定量 RT-PCR方法,并对引物浓度、退火温度和循环数进行优化,同时建立标准曲线,并将建立的荧光定量方法与常规RT-PCR方法比较。结果显示,本试验建立的荧光定量RT-PCR方法灵敏度可达1.0×100拷贝/μL,比常规 RT-PCR灵敏度高出至少100倍。特异性和重复性检测结果表明,该方法具有良好的特异性和较高的重复性。研究结果表明该方法可以对大熊猫轮状病毒进行稳定、可靠的检测,可以满足大熊猫轮状病毒特性研究和感染诊断的需要。

关键词: 大熊猫轮状病毒, SYBR GreenⅠ, 荧光定量RT-PCR

Abstract: This study aimed to develop an effective diagnosis for the giant panda rotavirus (GPRV) infection and early recessive infection of giant pandas, and to provide technical support for the quantitative analysis of the virus. Our study was based on the published sequence of the giant panda rotavirus strain vp4 gene in GenBank. A pair of specific primers was designed, and a fast accurate fluorescence quantitative RT-PCR detection method of giant panda rotavirus was established. Primer concentration, annealing temperature and the numbers of cycles then were optimized. In addition, standard curves were constructed and the Fluorescent Quantitative RT-PCR method and the routine RT-PCR were compared. The results show that the newly-developed Fluorescent Quantitative RT-PCR could detect 1.0×100copies/μL of plasmid DNA and its sensitivity was 100 times higher than that of the routine RT-PCR. The specificity and reproducibility test showed that the new method had the advantages of specificity and reproducibility. These results show that the method can be a stable and reliable detection method for GPRV, and could be used for the studies on characteristics of GPRV and for the diagnosingGPRV infection.

Key words: GPRV, SYBR GreenⅠ, FQ-RT-PCR