Taq Man探针实时荧光PCR方法检测黑蜂王台病毒的方法建立和应用  

Establishment of method and application for using a Taq Man probe- based Real- time fluorescence quantitative PCR method to detect black queen cell virus

作　　者：杨倩[1] 宋战昀[2] 石建平[2] 张健[3] 王振国[2] 孟庆峰[2] 崔焕忠[1] 王全凯[1,4] 李敏思[5] 郑言[1] 

机构地区：[1]吉林农业大学,长春130118 [2]吉林出入境检验检疫局,长春130062 [3]长春生物制品研究所有限责任公司,长春130062 [4]吉林省中韩动物科学研究院,长春130600 [5]甘肃农业大学,兰州730070

出　　处：《黑龙江畜牧兽医》2015年第4期39-42,247,共5页Heilongjiang Animal Science And veterinary Medicine

基　　金：国家自然科学基金项目(31001065);国家质检总局科技计划项目(2013IK033);国际科技合作专项(2011DFA32900)

摘　　要：为了建立实时荧光PCR方法以检测蜜蜂黑蜂王台病毒,试验依据Taq Man荧光标记探针技术原理,针对蜜蜂黑蜂王台病毒编码衣壳蛋白的保守序列区域,设计出1对特异性引物和1条探针,建立了一种快速检测黑蜂王台病毒(BQCV)的荧光PCR方法,对2005年从12省收集到的18种蜜蜂病料进行检测,确定各地区感染BQCV情况。结果表明:该方法对蜜蜂黑蜂王台病毒有较好的特异性,与其他蜜蜂病毒之间均无交叉反应;检测灵敏度可达1.0×102拷贝/μL阳性质粒,可对低病毒含量的样品进行准确检测;变异系数为1.3%;应用该方法对2005年从12省收集到的18种蜜蜂病料进行检测,4 h内即可报告检测结果。说明该方法具有快速、灵敏、特异及重复性好等优点,适用于蜜蜂中黑蜂王台病毒的快速检疫。To establish a Real- time fluorescence PCR method to detect black queen cell virus（ BQCV）,contrary to the conserved sequence regions of the encoded capsid protein of BQCV,a pair of primers and a probe were designed based on the technical principle of the Taq Man fluorescence- labeled probe,and then a Taq Man- based Real- time fluorescence PCR method was developed for rapid detection of BQCV. The pathological materials collected from 18 honeybees of 12 provinces in 2005,were detected to determine the case of BQCV infection in each region using the PCR method. The results showed that the method had good specificity for black queen cell virus without any cross- reactions with other honeybee viruses. Its detection sensitivity for positive plasmid was up to 1. 0 × 102 copies / μL,and the samples with low virus content could be accurately detected,and the coefficient of variation was 1. 3%. The test results could be reported within 4 h when using the method to detect the pathological materials collected from 18 honeybees of 12 provinces in 2005. The results indicate that the method has advantages of being rapid,sensitive,specific with good repeatability,and is suitable for rapid quarantine of black queen cell virus from honeybees.

关 键 词：蜜蜂 黑蜂王台病毒(BQCV) TAQ Man探针 荧光PCR检测 快速检测 

分 类 号：S891[农业科学—特种经济动物饲养] R446[农业科学—畜牧兽医]