摘要
为进一步阐明鸭肠炎病毒(DEV)的致病机理,本试验建立检测鸭肠炎病毒核衣壳蛋白质(NP)的互作蛋白质(PKCI)基因荧光定量PCR方法。针对PKCI基因设计特异性引物,经PCR扩增目的基因构建重组质粒p MD18-T-PKCI,将其作为阳性标准品构建荧光定量PCR标准曲线,并对建立的荧光定量PCR方法进行重复性、特异性和敏感性试验。结果显示:标准曲线的线性关系为Y=-3.31x+42.00,相关系数为-1.00,扩增效率为100%,熔解曲线仅出现单特异峰,对H5亚型、H7亚型和H9亚型禽流感病毒及新城疫病毒、鸭肝炎病毒均未检测到荧光信号。表明本试验建立的方法具有良好的稳定性、特异性和灵敏性。
To identify the pathogenesis of duck enteritis virus（ DEV）, a real-time fluorescent quantitative PCR assay for detecting the interacting protein （PKCI） gene of nucleocapsid protein（NP） of duck enteritis virus was developed. With designed specific primer, the PKCI gene was amplified by PCR. The recombinanted plasmid of pMD18-T-PKCI was used as positive standard to construct a standard curve by real-time fluorescent quantitative PCR, Y=-3.31 x ＋42. 00, with the correlation coefficient of -1.00 and the amplification efficiency of 100%. Melting curve appeared a single specific peak. The fluorescence signal were not detected in H5, H7, H9 subtype avian influenza viruses, newcastle disease virus, and duck hepatitis virus. The real-time fluorescent quantitative PCR method established in this study exhibited good stability, speeificity and sensitivity.
出处
《江苏农业学报》
CSCD
北大核心
2015年第2期389-393,共5页
Jiangsu Journal of Agricultural Sciences
基金
国家自然科学基金项目(31260607)
贵州省重点实验室培育项目[黔科合计Z字(2011)4008号]
关键词
鸭肠炎病毒
核衣壳蛋白质
蛋白质激酶C抑制蛋白质
荧光定量PCR
duck enteritis virus
nucleocapsidprotein
protein kinase C inhibitor（PKCI）
real-time flu-orescent quantitative PCR
