【摘要】 本研究中,采用T aqM an方法,根据新城疫病毒F基因核苷酸序列,设计合成多对引物,在上游引物和下游引物之间设计多条探针,通过对引物、探针的筛选,反应条件的选择和优化,建立了检测活禽和禽产品中中强毒力新城疫病毒的荧光RT-PCR方法。经对10株倍比稀释的中强毒力新城疫病毒的尿囊液进行检测后,表明所建立的荧光RT-PCR方法的检测极限在10-5~10-7之间,略低于鸡胚病毒分离方法(10-8~10-9);对收集到的所有新城疫病毒株和常见禽类病毒(包括禽流感病毒H 5、H 9亚型、IBDV、IBV、K IBV、CAAV、鸭瘟病毒、鸭肝炎病毒)进行检测,结果表明建立的方法不能检出其他的常见禽类病毒,特异性良好。进一步用建立的荧光RT-PCR检测人工感染SPF肉鸡的组织脏器、咽喉及泄殖腔拭子及临床样品中的中强毒力的新城疫病毒,并同鸡胚分离结果比较,结果表明荧光RT-PCR的敏感性同鸡胚分离试验基本一致。由于荧光RT-PCR方法快速,从处理样品开始到出结果只需不到4小时,而且检测样品量大,这就充分显示了其快速、敏感、特异的优势。在强调口岸快速通关的今天,该方法的建立无疑为活禽或禽产品的快速检验检疫提供了有效的手段。更多还原

【Abstract】 In this study,a real-time RT-PCR（RRT-PCR） was developed to detect velogenic and mesogenic strains of Newcastle disease virus（NDV） in live poultry and its products using TaqMan procedure by the screening and optimization of multiple pairs of primers between up-and down-streams of NDVF gene,probes and reaction conditions.The detection limit of RRT-PCR for velogenic and mesogenic strains of NDV in allantoic fluid was 10^-510^-7,less sensitive than that of virus isolation（VI） in embryonated chicken eggs(10^-8～10^-9),which was determined by testing the infective allantoic fluid of 10 strains at 10-fold serial dilution.The results also clearly demonstrated the high specificity of developed RRT-PCR for the detection of velogenic and mesogenic strains of NDV by testing all collected NDV stains and other avian virus including subtype H5 and H9 of AIV,IBDV,IBV,KIBV,CAAV,DPV and DVHV.Furthermore,results from comparative studies of RRT-PCR and VI using the different kinds of tissue homogenates,tracheal and cloacal swabs,which were collected from the broilers experimentally infected by NDV,and using clinical samples,showed the similar sensitivity between RRT-PCR and VI.in examination of NDV in tissues and tracheal and cloacal swabs.RRT-PCR was specific and rapid,and it only takes 4 hours from sample processing to obtaining results,suitable for screening samples in large scale.So it is advantageous to the exaltation of detection efficiency,especially in nowdays when fast detection in ports is highly emphasized.Therefore,this study has great significance for the entry-exit quarantine of poultry and its products.更多还原