禽流感H5、H7、H9亚型多重实时荧光RT-PCR检测试剂盒灵敏性试验  

Sensitive test of the multiplex real-time reverse transcriptase PCR kit for the rapid detection of subtype H5,H7 and H9 avian influenza virus

作　　者：秦智锋[1] 吕建强[1] 肖性龙 张东林[1] 李环玲[1] 陈书琨[1] 钟安清[1] 黄运生[1] 林庆燕[1] 林镜中 刘胜利[1] 

机构地区：[1]深圳出入境检验检疫局 [2]深圳太太基因工程有限公司,广东深圳518057

出　　处：《中国预防兽医学报》2006年第3期336-340,共5页Chinese Journal of Preventive Veterinary Medicine

基　　金：深圳出入境检验检疫局科学研究与技术开发重大项目

摘　　要：为了确定深圳出入境检验检疫局和深圳太太基因工程有限公司联合研制的禽流感H5、H7、H9亚型多重实时荧光RT-PCR检测试剂盒对H5和H9亚型禽流感的灵敏度,通过测定已知鸡胚半数致死量(ELD50)的标准毒株的进行了定量。利用9～11日龄SPF鸡胚,对深圳出入境检验检疫局保存的H5和H9亚型标准禽流感毒株进行了鸡胚半数致死量(ELD50)的测定,结果为H5亚型禽流感毒株的半数致死量为10-7.75/0.2mL,H9亚型禽流感毒株的半数致死量为10-8.5/0.2mL。用所研制的禽流感H5、H7、H9亚型多重实时荧光RT-PCR检测试剂盒对H5和H9亚型标准禽流感毒株原液进行10倍连续稀释,进行实时荧光RT-PCR灵敏度的测定,结果发现:试剂盒检测H5亚型标准禽流感毒株的灵敏度为标准毒株10-5倍稀释液,该稀释液含有281ELD50病毒量;检测H9亚型标准禽流感毒株的灵敏度为标准毒株10-8倍稀释液,该稀释液含有15.8ELD50病毒量。同时检测H5和H9时,试剂盒的灵敏度要比单独检测一个亚型时的灵敏度增加一个数量级。本灵敏度已经能够检测到感染鸡的喉拭子和(或)泄殖腔拭子所采集的病毒量,能够满足检验检疫的实际需要。In order to confirm the sensitivity of multiplex real-time reverse transcfiptase PCR （RRT-PCR） kit for the rapid detection of subtype H5 , H7 and H9 avian influenza virus, which developed by Shenzhen CIQ and Shenzhen Taitai genomics incorporation, a quantitative method was adopted to detect the embryo median lethal dose of H5 and H9 standard strains stocked in SZCIQ. the ELDs0 result of H5 strain was 10^-7.75/0.2 mL and H9 was 10^-8.5/0.2 mL. The multiplex real-time RT-PCR kit had a detection limit of 10.5 fold dilution of H5 standard strain and can detect 281 50 % egg lethal dose of this virus; and the kit had a detection limit of 10-7 fold dilution of H9 standard strain and can detect 15.8 50 % egg lethal dose of this virus. If to detect H5 strain and H9 strain simultaneously, the sensitivity would be ten fold of the single detection limit. Overall the sensitivity of the kit could detect the amount of virus collected by tracheal and cloacal swabs, and could satisfy the demand of quarantine in bureau.

关 键 词：禽流感病毒 鸡胚半数致死量 多重实时荧光RT-PCR 灵敏性试验 

分 类 号：S852.65[农业科学—基础兽医学] S855.3[农业科学—兽医学]