新城疫病毒通用型实时RT-PCR检测方法的建立与应用

采用TaqMan方法,经引物和探针的设计、筛选及反应条件优化,研究了检测活禽和禽产品中新城疫病毒的通用型实时RT-PCR(RRT-PCR)方法。结果显示,对12株分别为速发型、中发型、缓发型和疫苗株新城疫病毒的尿囊液倍比稀释液的检测极限在10-5~10-7之间;建立的方法与常见禽类病毒无交叉反应,特异性良好;在检测人工感染肉鸡的脏器组织、咽喉、泄殖腔拭子中病毒的灵敏度同鸡胚分离试验基本一致;弱毒疫苗免疫鸡群在免疫后14 d,应用本方法不能从咽喉、泄殖腔拭子中检测到病毒;临床样品检测表明,该方法不仅可以检出中强毒力新城疫毒株,也可检出缓发型野毒株和疫苗毒株。

Development and Application of Real-time RT-PCR for Detection of Newcastle disease virus

To develop a rapid detecting method of Newcastle disease virus(NDV),a real-time RT-PCR(RRT-PCR) was established to detect various strains of Newcastle disease virus(NDV) in live poultry and its products using Taq Man procedure by designing primers and probes,screening and optimization of primers,probes and reaction conditions.The detection limit for 12 strains(velogenic,mesogenic,lentogenic and vaccine strain) of RRT-PCR was 10~(-5)~10~(-7),which was determined by testing the infective allantoic fluid at 10-fold serial dilution.The results also clearly demonstrated the high specificity of developed RRT-PCR for the detection of various strains of NDV by testing all collected other avian virus.Furthermore,comparative detection of RRT-PCR and virus isolation in embryonated eggs was carried through for the tissues homogenates,tracheal and cloacal swabs sampled from the broilers infected by NDV,which suggested the similar sensitivity between them.By detecting the tracheal and cloacal swabs taken from vaccinated broilers,the result showed negative at 14 day post vaccination.Not only were the developed method used to detecting the velogenic and mesogenic stains,but also lentogenic and vaccine stains by detecting clinical samples.