鸽圆环病毒TaqMan荧光定量PCR检测方法的建立及初步应用
Establishment and application of PCR assay for detect of pigeon circovirus
郭学波 1赵承锋 2吴瑶 1吴琼 1胡学文1
作者信息
- 1. 汉唐和元(武汉)科技有限公司,湖北武汉 430070
- 2. 安陆市动物疫病预防控制中心,湖北孝感 432600
- 折叠
摘要
为建立快速灵敏检测鸽圆环病毒(PiCV)的方法,参考GenBank上给出的中国流行PiCV毒株序列(登录号:OL901205.1),合成了 PiCV的全基因组重组质粒,并依据PiCV的Rep基因保守序列设计多组引物、探针,从中优选出1组扩增效果最佳的引物、探针,建立TaqMan荧光定量PCR检测方法.结果显示,该方法具有良好的灵敏性,使用合成的标准重组质粒进行试验,最低检测限为21 copies/μL,且在标准质粒浓度2.1 × 108～2.1 × 10 copies/μL范围内具有良好的线性关系,线性相关系数 R2=0.999 1;该方法具有高度的特异性,仅与PiCV发生特异性反应,不与鸽疱疹病毒1型、鸽腺病毒、新城疫病毒及鸽轮状病毒发生交叉反应;该方法的重复性亦佳,批间变异系数小于2％,批内变异系数低于1％.使用本方法检测50份疑似病料咽拭子样本,样本阳性检出率为94％,高于普通PCR的82％,两者符合率为100％.综上所述,建立的PiCV TaqMan荧光定量PCR方法可用于PiCV的临床检测.
Abstract
To establish a method for the rapid and sensitive detection of pigeon circovirus(PiCV),a genome-wide recombinant plasmid of PiCV had been constructed according to a PiCV popular strain genome sequence(GenBank:OL901205.1).Subsequently,multiple sets of primers and probes were designed based on the conserved sequence of PiCV Rep gene.After screening,the optimized primers and probes com-bination which presented the best amplification efficiency was selected.And the TaqMan fluorescent quantitative PCR detection method was established.To test the sensitivity of this method,the synthet-ic standard recombinant plasmid was employed.The results showed that the minimum detection limit of template is 21 copies/μL and the data can keep a good linear relationship over the range 2.1 × 101-2.1 × 108 copies/μL with the linear correlation coefficient R2=0.999 1.This method also show a highly specificity to PiCV which has no signal can be detected from pigeon herpesvirus type 1,pigeon aden-ovirus,Newcastle disease virus and pigeon rotavirus.The repeatability of the method is also excel-lent.The coefficient of variation is less than 2％,and the intra-assay coefficient of variation is less than l％.By using 50 clinical samples,the positive detection rate of this method is 94％while that of PCR is 82％.The coincidence rate of the two methods was 100％.In summary,the TaqMan fluorescent quantitative PCR detection method of PiCV established in this experiment can be used for clinical de-tection of PiCV.
关键词
鸽圆环病毒/TaqMan荧光定量PCR/检测/应用Key words
pigeon circovirus/TaqMan fluorescence PCR/detection/application引用本文复制引用
出版年
2024
国家科技期刊平台
NSTL
万方数据