恒温实时荧光法快速检测简单异尖线虫方法的建立
A real-time fluorescence loop-mediated isothermal amplification method for the detection of Anisakis simplex
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摘要: 为了快速鉴定简单异尖线虫,本研究建立了一套检测简单异尖线虫DNA的恒温实时荧光环介导等温扩增（loop-mediated isothermal amplification,LAMP）方法,根据LAMP方法原理,针对简单异尖线虫ITS2区域设计引物特异性识别靶标基因。进行了特异性、灵敏度、重复性和实际样品的测试,并与传统的PCR方法进行比较。结果表明,该方法能够特异性扩增简单异尖线虫DNA,对含有简单异尖线虫ITS2目的基因片段的质粒DNA检测限为1 fg/μL,灵敏度比传统的PCR方法高100倍,重复性良好,对实际样品进行检测,与传统的PCR测序方法结果相符。本研究建立的恒温实时荧光快速检测方法适用于特异性检测简单异尖线虫。Abstract: We established a real- time fluorescence loop- mediated isothermal amplification( LAMP) approach for the sensitive,rapid and reliable detection of Anisakis simplex.The method was based on LAMP reaction.Three sets of the specificity and sensitivity of primers were designed from ITS2 r DNA. The specificity and sensitivity were tested.Specific amplification products were obtained with A.simplex,while no amplification products were detected with DNA of related parasites,demonstrating the specificity of the assay.The capable detection of plasmid DNA in the method was 1 fg / μL.The real- time fluorescence LAMP assay was proved to be 100 times more sensitive than a conventional polymerase chain reaction for detection of A. simplex. Samples testing by the real- time fluorescence LAMP method and PCR method showed that,the result of the LAMP method was the same with PCR method.The established real- time fluorescence LAMP method was suitable for specifically detecting A.simplex.
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