环介导等温扩增联合核酸层析试纸快速鉴定燕窝基原

Rapid Identification of Edible Bird′s Nest by Loop-Mediated Isothermal Amplificatio(LAMP) Combined with a Nucleic Acid Lateral-Flow Device(LFD)

目的:应用环介导等温扩增技术(LAMP)联合核酸层析试纸(LFD),基于燕窝中残留的线粒体DNA(mtDNA)建立燕窝基原的鉴定方法。方法:以不同生物基原的燕窝样品及伪品为研究对象,提取mtDNA,设计LAMP引物,建立LAMP扩增方法,并评价LAMP扩增浊度法检测与恒温水浴LAMP扩增联合LFD检测方法的特异性及灵敏度。结果:当反应温度为63℃时,燕窝线粒体中的细胞色素基因(mtDNA-cytb)的扩增效果最佳,而且LAMP扩增浊度检测与恒温水浴LAMP扩增联合LFD检测表现出一致的结果,均能有效区分燕窝样品的基原及伪品;在4.32×10~8～4.32×10~3 copies/μL的质粒标准品中具有良好的扩增检测效果。结论:LAMP扩增联合LFD的检测方法可以检测出燕窝核酸成分,对燕窝的基原鉴定具有良好的特异性和灵敏度,用于燕窝的检测快速准确,灵敏简便。

Objective:To identification edible bird′s nest(EBN)by loop-mediated isothermal amplification(LAMP)combined with nucleic acid chromatography strip(LFD),based on residual mitochondrial DNA(mtDNA).Methods:Different biologic original edible bird′s nest and adulterant were extracted for the nuclear acid.LAMP primers for establishing LAMP amplification method were designed,meanwhile the specificity and sensitivity of LAMP amplification by turbidity detection and LAMP amplification in constant temperature water bath combined with LFD were evaluated.Results:When the reaction temperature was at 63 ℃,the amplification of mtDNA-cytb of EBN was the best.The detection of LAMP amplification by turbidity was similar with the result of LAMP amplification combined with LFD,which could effectively distinguish biological basis of the EBN with the counterfeit goods.In addition,the plasmid standard at 4.32×10^8~4.32×10^3 copies/μL had a good amplification.Conclusion:The method of LAMP combine with LFD can detect the nucleic acid composition of EBN and has good specificity and sensitivity for the detection.It can be used for rapid,specific,sensitive and simple identification of EBN.