吉林大学学报(理学版)

• 生命科学 • 上一篇    

猫杯状病毒荧光定量PCR检测方法的建立及初步应用

姜雪1, 高玉伟2, 胡桂学1, 杨松涛2, 赵艳丽1, 刘秋燕1, 徐春忠3, 梁秀娟4, 夏咸柱2   

  1. 1. 吉林农业大学 动物科学技术学院, 长春 130118; 2. 解放军军事医学科学院 军事兽医研究所, 长春 130062;3. 上海野生动物园, 上海 201300; 4. 吉林省野
    生动物救护繁育中心, 长春 130122
  • 收稿日期:2012-12-03 出版日期:2013-09-26 发布日期:2013-09-17
  • 通讯作者: 胡桂学 E-mail:huguixue901103@163.com

Development and Preliminary Application of RealTime  PCR Assayfor Detection and Quantization of Feline Calicivirus

JIANG Xue1, GAO Yuwei2, HU Guixue1, YANG Songtao2, ZHAO Yanli1,LIU Qiuyan1, XU Chunzhong3, LIANG Xiujuan4, XIA Xianzhu2   

  1. 1. College of Animal Science and Technology, Jilin Agricultural University, Changchun 130118, China;2. Institute of Military Veterinary, Academy of Military Medical Sciences, Changchun 130062, China;3. Shanghai Wild Animal Park, Shanghai 201300, China;4. Jilin Wild Animal Conservation and Breeding Center, Changchun 130122, China
  • Received:2012-12-03 Online:2013-09-26 Published:2013-09-17
  • Contact: HU Guixue E-mail:huguixue901103@163.com

摘要:

根据GenBank中编码猫杯状病毒(FCV)衣壳蛋白ORF2的保守序列, 设计并合成了一对引物和相应的TaqMan探针, 建立了快速检测FCV的荧光定量PCR
方法. 通过对该方法的反应体系和反应条件进行优化, 建立了标准曲线, 给出了特异性、 敏感性和重复性实验, 并对临床24份疑似样品(其中阳性3份、 阴性21份)进行检测.  结果表明: 该方法检测cDNA的线性关系为0.992, 线性范围为2.26×1011~2.26×101拷贝/μL; 可检测出样品中的FCV, 其他猫相关病毒检测为阴性; 批内重复性实验变异系数为2.158%, 与病毒分离结果相符.

关键词: 猫杯状病毒(FCV), 荧光定量PCR, 检测方法

Abstract:

To detect feline calicivirus (FCV), a realtime quantitative PCR (qPCR) assay was established by using a pair of primers and TaqMan probe designed and synthesized according to the conserved capsid protein gene ORF2 sequence of FCV in the GenBank. The reaction system and conditions of the qPCR assay were optimized and the standard curve was established. Further, the specificity, sensitivity and repeatability test were also assessed. The established quantitative PCR assay was applied to detecting clinical samples infected by FCV. The results show that the correlation rate of the standard curve for the qPCR was 0.992. The detected quantity was from 2.26×101 to 2.26×1011 copies/μL of FCV cDNA. With the qPCR method, a reliable diagnostic result was obtained for detecting FCV samples. But detection of other feline pathogenic agents was negative. In addition, reproducible assay showed a good performance of repeatability and the variation coefficient of intragroup was 2.158%.

Key words: feline calicivirus(FCV), flurogenic quantitative polymerase chain reaction, detection method

中图分类号: 

  •