B亚型禽偏肺病毒RT-LAMP快速检测方法的建立与应用  

Establishment and application of RT-LAMP system for rapid detection of avian metapneumovirus subgroup B

作　　者：鞠小军[1] 刁有祥[1] 唐熠[1] 武利利[1] 薛聪[1] 崔京腾[1] 宋晓娜[1] 

机构地区：[1]山东农业大学动物科技学院,山东泰安271018

出　　处：《中国兽医学报》2013年第6期813-817,共5页Chinese Journal of Veterinary Science

基　　金：山东省科技发展计划项目(2010GNC10912)

摘　　要：利用Primer Explorer V4在线软件设计针对B亚型禽偏肺病毒(aMPV)F基因的特异性引物,并从反应时间、温度、各组分浓度等方面优化了反应体系和反应条件,建立了B亚型aMPV逆转录环介导等温核酸扩增(RT-LAMP)快速检测方法。该方法能够在63℃条件下1h内实现B亚型aMPV F基因片段的特异性扩增,与其他病毒,如H9N2亚型禽流感病毒(AIV)、新城疫病毒(NDV)、传染性支气管炎病毒(IBV)、传染性法氏囊病病毒(IBDV)等的核酸无交叉反应。反应结果可直接用肉眼判断。对质粒DNA的最小检测量为1×102拷贝/μL。利用建立的检测方法对20份疑似aMPV样品进行检测,其阳性检出率为10%。结果表明,建立的B亚型aMPV RT-LAMP检测方法具有快速、准确、特异性强、灵敏度高的特点,可应用于相关疾病的临床诊断。A rapid diagnostic method of reverse transcription loop-mediated isothermal amplification （RT-LAMP） for avian metapneumovirus（aMPV） subgroup B was established with a set of special primers designed for aMPV F gene with Primer Explorer V4. And the reaction system and reaction conditions were optimized including the reaction time, temperature, concentration of various com- ponents and other aspects. The fragment of avian metapneumovirus（aMPV） subgroup B F gene could be specifically amplified by the RT-LAMP at 63~C within 1 h and the amplification results of H9N2 avian influenza virus（AIV）, Newcastle disease virus（NDV）, infectious bronchitis virus （IBV） ,infectious bursal disease virus（IBDV） and soon were negative. The amplification products could be observed by naked-eye. The detection limit of the system was found to be 1 X 10Zcopies/ /~L plasmid sample. The positive rate of twenty chicken samples suspected ampv was 10~. The re- sults showed that the RT-LAMP diagnostic method for aMPV subgroup B appeared to be fast,ac- curate, highly sensitive and specific and it could be applied to the clinical diagnosis of the relevant disease.

关 键 词：B亚型禽偏肺病毒 逆转录环介导等温核酸扩增 快速检测 

分 类 号：S852.65[农业科学—基础兽医学]