一种可视化双重荧光RT-LAMP方法鉴别不同毒力新城疫病毒

Development and evaluation of a duplex fluorescence loop-mediated isothermal amplification method for distinguishing detection of virulent and non-virulent NDV

环介导等温扩增(loop-mediated isothermal amplification,LAMP)检测技术具有快速、敏感、仪器简单的优势。为了达到多重鉴别检测的目的,将分子信标引入LAMP反应体系,建立一套鉴别不同毒力新城疫病毒(Newcastle disease virus,NDV)的可视化双重荧光RT-LAMP方法。分别设计2套扩增不同毒力ClassⅡNDV F基因的LAMP引物,针对区分NDV毒力的分子标志F蛋白裂解位点的基序差异设计2条分子信标。中、强毒NDV分子信标5′端标记FAM荧光基团,3′端标记Dabcyl淬灭基团;弱毒NDV 5′端标记CY5荧光基团,3′端标记BHQ3淬灭基团。用经过条件优化的RT-LAMP体系对不同毒力NDV及其他病原进行鉴别检测,结果显示,LAMP引物能特异地扩增ClassⅡNDV,在荧光成像仪下,中、强毒NDV显示绿色荧光,弱毒显示红色荧光,混合感染显示黄色荧光,而其他家禽常见病原无荧光显示;以体外转录的NDV ssRNA片段为模板,建立的RT-LAMP反应体系最低检测限度为100 copies/μL;随机检测疑似NDV病料20份,鉴定结果与经分离、测序鉴定的结果一致。本研究建立的分子信标与LAMP结合的检测方法,可为NDV多重检测提供新的思路。

Loop-mediated isothermal amplification(LAMP)is a reliable and rapid sequence-specific isothermal nucleic acid amplification technique.Here,a visual dual-fluorescence RT-LAMP method in conjunction with molecular beacons for distinguishing virulent and non-virulent NDV was developed.Two sets of LAMP primers were designed for amplifying F gene of different virulence ClassⅡNDV.Two molecular beacons were designed based on the DNA sequence difference of cleavage sites of F protein which was the molecular marker for distinguishing NDV virulence.The virulent NDV beacon was labeled with fluorescence group FAM at 5′end and quenching group Dabcyl at 3′end.The non-virulent NDV beacon was labeled with fluorescence group CY5 at 5′end and quenching group BHQ3 at 3′end.Different virulence NDV were detected with the optimized dual-fluorescence RT-LAMP assay,the results showed that only ClassⅡNDV could be specifically amplified,under the fluorescence imager,virulent NDV showed green fluorescence,non-virulent NDV showed red fluorescence,mixture showed yellow fluorescence,and no other pathogens of poultry were detected.The detection limits of NDV were determined to be 100 copies/μL by using in vitro transcribed NDV ssRNA as the template.Twenty suspected clinic samples were randomly detected with this duplex RT-LAMP assay,the results were consistent with those identified by isolation and sequencing.The molecular beacon combined with RT-LAMP established in this study can provide a new idea for multiplex detection.