RT-LAMP 及RT-PCR 方法快速检测玉米褪绿斑驳病毒的比较与应用

    Comparison and application of RT-LAMP and RT-PCR methods for MCMV detection

    • 摘要: 利用玉米褪绿斑驳病毒外壳蛋白基因为靶标序列设计引物,通过优化反应温度、引物浓度、反应时间以及评价环引物效果,建立了检测玉米褪绿斑驳病毒反转录环介导等温扩增( RT-LAMP) 方法,并与RT-PCR 方法比较。结果表明,RT-LAMP 在64℃等温条件下反应45 min,最低可检测到280 fg 的目的片段,灵敏度是RT-PCR 的100 倍。以甘蔗花叶病毒、玉米矮花叶病毒、小麦线条花叶病毒、玉米白线花叶病毒及玉米褪绿斑驳病毒等5 种病毒为检测对象,证明该方法针对玉米褪绿斑驳病毒具有很强的特异性。利用RT-LAMP 方法对100 份模拟病毒感染的玉米种子进行检测,病毒检出率为100%。

       

      Abstract: For the detection of Maize chlorotic mottle virus(MCMV),a reverse-transcription,loop mediated isothermal amplification( RT-LAMP) assay with high sensitivity and specificity was established. A set of five primers were designed,based on the coat-protein sequence of MCMV. By optimizing the concentration of primers,the best temperature,the suitable reaction time,and loop primer,the RT-LAMP was built by incubation at 64℃ for only 45 min with loop primer. The detection limit of RT-LAMP assay was 280 fg of MCMV RNA,which was 100 times more sensitive than that of RT- PCR assay. High species-specificity of RT- LAMP method was confirmed by the assay of 5 pathogens such as MCMV,SCMV,MDMV, WSMV and MWLMV. In addition,all the simulated samples were detected by LAMP assay,detection rate was 100%.

       

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