One-step reverse transcription loop-mediated isothermal amplification assay for rapid detection of melon yellow spot virus

被引:9
作者
Zeng, Rong [1 ]
Xu, Li-hui [1 ]
Gao, Shi-gang [1 ]
Ni, Xiu-hong [2 ]
Chen, Chun-lei [2 ]
Chen, Jian-cai [2 ]
Dai, Fu-ming [1 ]
机构
[1] Shanghai Acad Agr Sci, Inst Plant Protect, Shanghai Key Lab Protect Hort Technol, Shanghai 201403, Peoples R China
[2] Pudong Dist Agrotechnol Extens Ctr, Shanghai 201300, Peoples R China
关键词
MYSV; RT-LAMP; Optimization; Visualization; TOSPOVIRUS; WATERMELON; THAILAND; JAPAN;
D O I
10.1007/s10658-015-0821-6
中图分类号
S3 [农学(农艺学)];
学科分类号
0901 ;
摘要
A one-step, accelerated reverse transcription loop-mediated isothermal amplification (RT-LAMP) procedure was developed for the detection of Melon yellow spot virus (MYSV). The four primers required for accelerated RT-LAMP were designed to anneal onto a conserved region in MYSV N gene. Reaction temperature, optimal Mg2+ concentration, and the ratio of outer/inner primers affected reaction efficiency. MYSV RT-LAMP optimal reaction parameters were obtained in this study. The RT-LAMP was specific and sensitive in detecting MYSV in 60 minutes, with a detection limit of 0.5 x 10(-4) mu g total RNA per reaction, and a ladder-like pattern observed after agarose gel electrophoresis. RT-LAMP was 100-fold more sensitive than the conventional one-step reverse transcription polymerase chain reaction (RT-PCR) using serial dilutions of total RNA. RT-LAMP was a specific, sensitive, and low cost diagnostic tool that should be of value in more accurate determination of the distribution and host range of MYSV.
引用
收藏
页码:119 / 124
页数:6
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