摘要
本研究结合非对称RT-PCR和基因芯片两种技术,构建可同时区分AIV的H5、H7、H9血凝素亚型和N1、N2神经氨酸酶亚型以及鸡传染性支气管炎病、新城疫病、鸡传染性法氏囊病的基因芯片。分别T/A克隆部分禽流感病毒的M基因,H5、H7、H9亚型HA基因,N1、N2亚型NA基因,鸡传染性支气管炎病毒np基因、新城疫病np基因、鸡传染性法氏囊A基因,并构建重组质粒;以重组质粒为模板,用PCR方法扩增制备探针,纯化后点于氨基修饰的载玻片上,制备基因芯片;常规方法从待检样品中提取RNA,用Cy3标记引物进行RT-PCR,将荧光素引入PCR产物中;并对扩增条件进行优化。研究发现检测探针可特异性的与相应的标记样品进行杂交,呈现较强的杂交信号;该方法可以准确进行4种主要禽病的鉴别诊断和禽流感主要流行亚型鉴定,对感染样品和现地样品检测结果与RT-PCR、鸡胚接种有较高一致性,符合率分别为100%和96%。结果显示该方法可用于同步鉴别禽流感、鸡传染性支气管炎、新城疫、鸡传染性法氏囊病,以及现地主要流行的禽流感亚型,是一种有效的新方法。
A new method of detecting H5, H7, H9, N1, N2, M genes of AIV, and IBV, NDV, IBDV was developed using dissymmetry PCR and DNA microarray methods. The HS, H7, H9, N1, N2, M genes of AIV, np gene of IBV, np gene of NDV, and A gene of IBDV, were cloned respectively, and respectives PCR products spotted on the amido-coating-glass slides as the probes to form a DNA microarray. Total RNA from samples were extracted and cDNA were synthesized using fluorescent Cy3-1abeled primer by Reverse transcriptase PCR. Strong signals of hybridization was obtained and the hybridization pattern was in consistence with the known grid location of each target. This new method can simultaneously detect AIV, IBV, IBDV, NDV and had a coincidence rate of 100 % and 96 % respectively with the egg inoculation and RT-PCR. In addition, it can identify the hemagglutinin subtypes H5, H7, H9 and neuraminidase subtypes N1, N2 of AIV.
出处
《中国预防兽医学报》
CAS
CSCD
北大核心
2008年第12期954-958,共5页
Chinese Journal of Preventive Veterinary Medicine
基金
国际合作项目(2007DFR30360)
关键词
不对称PCR
基因芯片
禽病
禽流感亚型
鉴别
dissymmetry PCR
microarrays
poultry disease
subtypes of AIV
detection
