新城疫病毒3种检测方法的研究比较

Comparison of Three Methods for Detection Newcastle Disease Virus

目的:利用3种方法对新城疫(Newcastle disease virus,NDV)病毒进行检测并对这3种检测方法的优缺点做出比较。方法:分别将NDV强毒F48E9和弱毒Lasota接种SPF鸡胚后,获取尿囊液。利用双抗夹心ELISA法、悬液芯片系统以及RT-PCR进行检测。通过对制备的针对新城疫病毒的抗体4D9和6C4蛋白浓度测定后,选择6C4进行生物素标记,将4D9作为固相捕获抗体,利用生物素-链霉亲和素放大系统构建双抗夹心检测体系。通过对Genebank上已发表的新城疫强弱毒F基因进行电脑分析后,设计一组针对NDV强弱毒的通用型引物,分别对强弱毒进行RT-PCR并检测其检出限。结果:ELISA法对NDV强弱毒尿囊液的检出灵敏度为1∶160,但操作繁琐,耗时长;液相芯片对强弱毒尿囊液的检出限为1∶160和1∶320,然而和ELISA相比,操作较为方便,但仪器设备昂贵。RT-PCR对强弱毒RNA检出限分别为259pg和14pg,与前两种方法相比,PR-PCR在核酸水平上对病毒进行检测,理论上灵敏度较高,但是所需试剂、设备昂贵,且实验人员还需一定的技能培训。

Newcastle disease viruses（NDV） are detected by three detecting tests and make a comparison of advantages and disadvantages about these tests.The allantoic fluid are obtained through inoculating the NDV virulent F48E9 and avirulent Lasota to SPF chicken embryo and detecting by double antibody sandwich ELISA,suspension array system and RT-PCR.After measure the concentration about antibody 6C4 and 4D9 against NDV,6C4 was selected as biotin labeled antibody,alternative 4D9 as solid phase capture antibody,the double antibody sandwich detection is based on biotin-streptavidin amplification.Design a group of universal primes against NDV after analyzing the F gene in NDV virulent and avirulent viruses which have been published on genebank and measure the detection limit by RT-PCR.The results show that the detection sensitivity of NDV virulent and avirulent viruses is 1∶160 by ELISA,but operation is cumbersome and time-consuming;the detection limit is 1∶160 and 1∶320 through suspension array system,respectively,however,its operation is more convenient and the equipments are expensive compared with ELISA.The detection limits of RNA in virulent and avirulent viruses are 259pg and 14pg,compared with previous two methods,RT-PCR is a measurement on nucleic acid level,it should have high sensitivity in theory,but the necessary reagents are considerable expensive and the laboratory personnel need training in certain skills.