摘要
【目的】建立鸭源新城疫病毒逆转录环介导等温核酸扩增(RT-LAMP)快速检测方法,为鸭源新城疫病毒的快速诊断提供支持。【方法】选取鸭源新城疫病毒NP基因的相对保守区序列,利用Primer Explorer V4在线软件设计了3对RT-LAMP引物,以鸭源新城疫病毒SDFCH株RNA为模板进行RT-LAMP反应,通过对反应温度、时间及反应体系各组分浓度的筛选,优化反应体系和反应条件,然后对该方法的灵敏性和特异性进行检测(以RT-PCR方法为对照),并将其应用于临床病料检测。【结果】优化的RT-LAMP反应体系能够在63℃下1h内实现目标核酸区段的大量扩增,反应结果可直接用肉眼判断;建立的鸭源新城疫病毒RT-LAMP检测方法特异性较强,灵敏度较高,与其他病毒,如H9N2亚型禽流感病毒(AIV)、鸭呼肠孤病毒(DRV)、禽偏肺病毒(aMPV)、传染性支气管炎病毒(IBV)等的核酸无交叉反应,可检测到1×10-3稀释度的目标RNA(0.1pg/μL),较普通RT-PCR的灵敏性高10倍;利用建立的鸭源新城疫病毒RT-LAMP检测方法对24份疑似鸭源新城疫样品的阳性检出率为41.7%。【结论】建立的鸭源新城疫病毒的RT-LAMP检测方法,具有快速、准确、特异性强、灵敏度高的特点。
【Objective】 The study was conducted to establish a rapid diagnostic method of reversetranscription loop-mediated isothermal amplification（RT-LAMP） for Newcastle disease virus（NDV） in ducks,and provide supports for the rapid detection of Newcastle disease.【Method】 Six special primers were designed for NDV NP gene with Primer Explorer V4.The RT-LAMP reaction was carried out taking the RNA of Newcastle disease virus SDFCH as its template.The reaction system and conditions were optimized by the sifting of temperature,time and the the concentration of all components.Then we assessed its specificity and sensitivity（take RT-PCR for contrast）,and applied it to the inspection of clinical material.【Result】 The Newcastle disease virus（NDV） RNA could be amplified by the optimized RT-LAMP at 63 ℃ within 1h and the amplification products could be observed under naked-eye.The diagnostic method was sensitive and specific,for the amplification results of H9N2 avian influenza virus（AIV）,duck reovirns（DRV）,Avian metapneumovirus（aMPV） and infectious bronchitis virus（IBV）were negative,and the detection limit of the system was found to be 1×10-3 RNA（0.1 pg/μL） sample,which was 10-fold higher than that of the traditional RT-PCR.The positive rate of twenty-four duck samples suspected newcastle disease was 41.7%.【Conclusion】 The RT-LAMP diagnostic method for Newcastle disease virus（NDV） in ducks was established and it appeared to be fast,accurate,highly sensitive and specific for the purpose.
出处
《西北农林科技大学学报（自然科学版）》
CSCD
北大核心
2011年第12期15-20,共6页
Journal of Northwest A&F University(Natural Science Edition)
基金
现代农业产业技术体系建设专项(nycytx-43-34)
公益性行业(农业)科研专项(201003012)
