摘要
为建立一种针对鸽圆环病毒(PiCV)的快速诊断方法,试验根据PiCV的Rep保守基因序列,设计合成一对特异性引物,建立了PiCV荧光定量PCR检测方法,对其敏感性、特异性、重复性进行评价并利用该方法对临床样本进行检测。结果表明:在最佳反应条件下,所建立的PiCV荧光定量PCR方法在1.43×10^(2)~1.43×10^(8)copies/μL标准品范围内具有良好的线性相关性,线性相关系数为0.998;敏感性试验结果显示,该方法最低可检测到1.43×10^(2)copies/μL标准品,是常规PCR检测方法的1000倍;特异性试验结果显示,该方法与其他常见的鸽病病原不发生交叉反应;重复性评价结果显示,批内与批间的变异系数均小于0.8%;所建立PiCV荧光定量PCR方法对临床鸽血清样品检测结果显示,PiCV阳性率达38.7%,高于常规PCR方法的检测阳性率(28.7%),说明该方法具有良好的适用性。研究结果PiCV的流行病学调查、病原学监测及定量研究奠定了基础。
To establish a rapid diagnosis method for pigeon circovirus(PiCV),this experiment designed and synthesized a pair of specific primers based on the Rep gene sequence of PiCV,and established a SYBR Green Ⅰ real-time PCR method for the detecting of PiCV.The sensitivity,specificity and reproducibility of the method were evaluated and the clinical samples were detected by this method.The results showed that under the best reaction conditions,the method had good linear relationship in the range of 1.43×10^(2)to 1.43×10^(8)copies/μL,and the correlation coefficient was 0.998.The sensitivity results showed the detection limit was 1.43×10^(3)copies/μL,which was 1000 times lower than that of ordinary PCR.The specificity results showed that this method had no specific amplification for PPMV-1,PiHV,FAdV,PCV2,pigeon Salmonella Typhimurium,Escherichia coli.The repeatability experiment results showed that the coefficient of variation between and within groups was less than 0.8%.The detection results of clinical pigeon serum samples showed that the established method had good applicability with the PiCV positive rate of 38.7%,which was higher than that of routine PCR method(28.7%).This method could be used for epidemiological investigations,etiological surveillance and quantitative studies for PiCV.
作者
亓玉卓
梁琳
朱亚丽
梁瑞英
贾亚雄
单虎
崔尚金
QI Yuzhuo;LIANG Lin;ZHU Yali;LIANG Ruiying;JIA Yaxiong;SHAN Hu;CUI Shangjin(Institute of Animal Science,Chinese Academy of Agricultural Sciences,Beijing 100193;Beijing Science Observatory Experimental Station for Veterinary Drugs and Diagnostic Technology,Ministry of Agriculture and Rural Affairs,Beijing 100193;College of Veterinary Medicine,Qingdao Agricultural University,Qingdao,Shandong 266109)
出处
《中国家禽》
北大核心
2021年第3期22-27,共6页
China Poultry
基金
家禽产业技术体系北京创新团队(BAIC04-2020)
中国农业科学院创新工程计划(ASTIP-IAS15)。
作者简介
亓玉卓(1996-),女,硕士研究生,研究方向为鸽传染病,E-mail:qyz0314@163.com;通讯作者:单虎(1967-),男,教授,主要从事动物传染病防治与生物制品学研究,E-mail:shanhu67@163.com;通讯作者:崔尚金(1971-),男,研究员,主要从事动物传染病病原学与流行病学研究,E-mail:cuishangjin@caas.cn。
