三种IBV检测方法比较研究  

Comparative study on three kinds of detection methods of avian infectious bronchitis virus

作　　者：潘龙[1] 李广兴[1] 聂思静 蔡皓璠[2] 李兰兰[1] 

机构地区：[1]东北农业大学动物医学学院,哈尔滨150030 [2]黑龙江职业学院农牧工程系,哈尔滨150080

出　　处：《东北农业大学学报》2014年第9期100-105,共6页Journal of Northeast Agricultural University

基　　金：黑龙江省自然科学基金项目(ZJN0702-01);国家自然科学基金项目(31172295;31272569)

摘　　要：IBV属于冠状病毒γ群,可引起鸡呼吸道疾病,对IBV诊断方法研究广泛,建立多种方法,而噬菌体展示技术广泛用于蛋白质研究中。使用分子生物学技术和噬菌体展示技术筛选到能与IBV S1蛋白特异性结合噬菌体建立三种IBV检测方法,Real-Time PCR、RT-PCR和噬菌体ELISA。结果表明,三种方法都可有效检测出传染性支气管炎病毒(IBV),证明三种方法可靠性,对这三种方法的灵敏性进行比较,敏感性上实时Real-Time PCR法最敏感,依次为RT-PCR和噬菌体ELISA技术,检测极限分别为7.64×101、7.64×103、1.21×105copies·μL-1,证明筛选的IBV S1蛋白特异性亲和噬菌体可用于IBV病毒诊断,具有较高特异性和敏感性,为IBV诊断提供新方法。Real-Time PCR、RT-PCR和噬菌体ELISA三种方法各具特点,可满足不同检测需要。Avian infectious bronchitis virus （IBV）, which belongs to group γ coronavirus, leads to chicken respiratory disease-infectious bronchitis （IB） and causes enormous economic loss. Latest researches on IBV diagnostic methods are undertaken very extensively, and rapid and specific diagnostic methods have obvious significance on the comprehensive prevention of IB. In this paper, molecular biology and phage display technique were used to establish three kinds of IBV diagnostic methods, i.e., real time quantitative polymerase chain reaction （qRT-PCR）, reverse transcription PCR （RT-PCR）, and phage-mediated enzyme-linked immunosorbent assay （phage-mediated ELISA）, and their characteristics were comparatively studied. The results showed that these three methods had the reliability to assay IBV specifical y, and they could detect IBV at least 7.64 × 101, 7.64 × 103, 1.21 × 105 copies·μL-1, respectively, in which qRT-PCR had the highest sensitivity. Furthermore, the high affinity phages to IBV S1 protein were employed with phage display technique to develop phage-mediated ELISA, and it had very good specificity and sensitivity for IBV diagnosis, and it is suitable to utilized veterinary clinic application.

关 键 词：IBV REAL-TIMEPCR RT-PCR 噬菌体ELISA 

分 类 号：S852.31[农业科学—基础兽医学]