摘要
本研究旨在建立一种快速、特异、敏感的H6亚型禽流感病毒(AIV)实时荧光定量RT-PCR检测方法。针对H6亚型AIV的血凝素(HA)基因序列保守区设计1对引物,并优化反应条件。结果表明,该方法的最低检测限为1.07×101拷贝质粒DNA,与常规PCR相比,灵敏度高出1 000倍;扩增产物的熔解曲线分析只出现1个单特异峰,无引物二聚体,Tm值为(80.81±0.08)℃,组内变异系数为0.08%～0.99%,组间变异系数为1.85%,可重复性好;对H1、H3、H5、H7、H9亚型禽流感病毒及新城疫病毒等其他呼吸道病原体无扩增;检测快速,从样本处理到报告结果仅需3.5 h。
The aim of this study is to develop a rapid, sensitive and specific Real-time RT-PCR （RRT-PCR） method for detection of H6 subtype avian influenza virus （AIV）. A pair of primers specific to the hemagglutinin （HA） gene was designed according to the HA gene se- quences of H6 subtype AIV available in GenBank, and the reaction conditions were optimized. The results showed that, the detection limit of RRT-PCR was 1.07×10^1 plasmid copies. The RRT-PCR was more sensitive than conventional PCR by 1000 folds. The melting curve analy- sis showed one specific peak at the melting temperature of （80. 81±0. 08 ）℃ without primer-dimers peak. The reproducibility of RRT-PCR was high and the coefficients of intra- and inter-assay assay variation were 0.08% -0. 99% and 1.85% , respectively. The specificity of the assay was high without any amplification for other viruses. In the RRT-PCR assay, the required time from sample processing to reporting of results was only 3.5h.
出处
《畜牧与兽医》
北大核心
2012年第11期12-16,共5页
Animal Husbandry & Veterinary Medicine
基金
广西科技项目(桂科攻10100014-5和桂科专项11-3)
广西特聘专家专项经费资助项目(2011B020)
国家百千万人才工程人选专项资金项目(No.945200603)共同资助
