应用二温式多重PCR技术鉴别鸡传染性支气管炎病毒和鸡传染性喉气管炎病毒

Detection of Infectious Bronchitis Virus and Infectious Laryngotracheitis Virus by Two-temperature PCR

关键词:  鸡传染性支气管炎病毒  鸡传染性喉气管炎病毒  二温式多重聚合酶链式反应  检测

Key words:  infectious bronchitis virus  infectious laryngotracheitis virus  two-temperature polymerase chain reaction  detection

摘要:

为了简化聚合酶链反应(PCR)程序和加快检测速度,将二温式PCR与多重PCR结合起来,建立一种同时检测鸡传染性支气管炎病毒(IBV)和鸡传染性喉气管炎病毒(ILTV)的二温式多重PCR技术。试验根据IBV和ILTV的基因文库,设计2对分别与IBV和ILTV某段基因序列互补的引物,用这2对引物对同一样品中的IBV、ILTV核酸模板进行二温式多重PCR扩增,结果均同时得到2条特异性大小与实验设计相符的1720bp(IBV)和647bp(ILTV)的扩增带,而对其他6种禽病病原的扩增结果均为阴性;敏感性测定结果表明,该二温式多重PCR技术能检出10 pg的IBV RNA模板和10 pg的ILTV DNA模板。

Abstract:

A two-temperature polymerase chain reaction was optimized to simultaneously detect two pathogens of IBV and ILTV for saving time of detection.Two sets of specific primers were designed according to the sequences of IBV and ILTV at the Gene Bank.All samples which contain IBV cDNA and ILTV DNA could be amplified by the two-temperature PCR using these two sets primers, yielding two specific bands of IBV 1720bp and ILTV 647bp. But no specific band amplified could be found from other six avian pathogenic viruses and bacteria.As little as 10pg of IBV RNA and 10pg of ILTV DNA were detected using gel electrophoresis in this two-temperature PCR.