鸡传染性支气管炎病毒Real-time RT-PCR检测方法的建立及应用

Development and Application of Real-time RT-PCR Assay for Chicken Infectious Bronchitis Virus

为定量检测鸡传染性支气管炎病毒(IBV)载量,建立IBV的Real-timeRT-PCR方法。用RT-PCR方法扩增出IBV的N基因片段,并克隆到pEASY-T3载体中,构建成含有N基因片段的重组质粒。应用该重组质粒进行SYBRGreenⅠReal-timePCR,建立了定量检测IBV核酸的标准曲线与直线回归方程,该方法显示:特异性强,检测下限至少达到5.58×102拷贝/μL,其重复性试验的变异系数小于3.2%;用建立的方法对实验接种IBVM41株的雏鸡组织中的病毒核酸进行了定量检测,检测结果显示:攻毒后肾脏中IBV含量高于支气管和肺脏,支气管和肺脏中病毒含量在攻毒后第3天高于攻毒后第7、10天,并证实临床表现与病毒载量之间存在密切关系。研究结果表明,建立的Real-timeRT-PCR检测方法特异性强、灵敏度高、可重复性好,可用于IBV的定量检测。

The objective of this study was to develop a Real-time RT-PCR assay of chicken Infectious Bronchitis Virus （IBV） and therefore to detect the load IBV quantitatively.A fragment of N gene in IBV was amplified with RT-PCR method,and was cloned into the vector pEASY-T3.Then,the recombinant plasmid containing the N gene fragment was constructed.The standard curve and corresponding linear regression equation of IBV nucleic acid level were developed by Real-time PCR based on SYBR Green I with the recombinant plasmid.This method showed a high specificity and had a detection limit of 5.58×10 2 copies/μL,and its coefficient of variations was less than 3.2% in the reproducible assays.The virus nucleic acid in tissue samples from chickens inoculated experimentally with IBV M41 strain was quantitatively determined with the established Real-time RT-PCR.The detection results showed that the load of IBV in the kidney was more than that in bronchus and lung after inoculation,and the virus load in the bronchus and lung on 3 days post-inoculation （DPI） were higher than those on 7 DPI and 10 DPI.Moreover,the correlation between the clinical manifestations and viral load was confirmed.The results indicated that this Real-time RT-PCR assay was of high specificity,sensitivity and reproducibility,and could be used for the quantitative detection of IBV.