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

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45min,最低可检测到280fg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%。...

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Published in广东农业科学 Vol. 44; no. 7; pp. 83 - 90
Main Author 单长林 周圆 李孝军 杨赛军 邵炜冬
Format Journal Article
LanguageChinese
Published 舟山出入境检验检疫局,浙江 舟山,316021 2017
Subjects
RT-PCR
反转录环介导等温扩增方法
检测
玉米褪绿斑驳病毒
反转录环介导等温扩增方法
玉米褪绿斑驳病毒
RT-PCR
检测
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ISSN1004-874X

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Summary: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45min,最低可检测到280fg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%。
Bibliography:44-1267/S
SHAN Chang-lin, ZHOU Yuan, LI Xiao-jun, YANG Sai-jun, SHAO Wei-dong （ Zhoushan Entry-exit Inspection and Quarantine Bureau, Zhoushan 316021, China ）
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 of5 pathogens such as MCMV,SCMV,MDMV, WSMV and MWLMV. In addition,all the simulated samples were detected by LAMP assay,detection rate was 100%.
MCMV; RT-LAMP; RT-PCR; detection
ISSN:1004-874X