单长林,周 圆,李孝军,杨赛军,邵炜冬.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[J].广东农业科学,2017,44(7):83-90 |
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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 |
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DOI:10.16768/j.issn.1004-874X.2017.07.014 |
中文关键词: 玉米褪绿斑驳病毒 反转录环介导等温扩增方法 RT-PCR 检测 |
英文关键词: MCMV RT-LAMP RT-PCR detection |
基金项目:舟山市科技计划项目(2015C31051) |
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中文摘要: |
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%。 |
英文摘要: |
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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