Ullah Parvaiz, Rashid Shahjahan, Wani Sumiah, Iralu Nulevino, Nabi Sajad Un, Ali Gowhar, Shikari Asif B, Hamid Aflaq
DNA Fingerprinting and Advanced Plant Virology Laboratory, AICRP NSP, Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir, Shalimar, Srinagar, Jammu Kashmir 190025 India.
ICAR-Central Institute of Temperate Horticulture, Rangreth Srinagar, Jammu Kashmir 191132 India.
Virusdisease. 2025 Mar;36(1):60-67. doi: 10.1007/s13337-025-00916-y. Epub 2025 Mar 28.
Bean common mosaic virus (BCMV) is one of the most serious and devastating of leguminous crops. In mung bean (), BCMV is an emerging virus causing enormous losses to the crop, thereby reducing the production and profitability of the crop. Being seed borne and aphid transmitted virus, it important to reduce the spread and prevent its transfer to new geographical locations using rapid, specific and sensitive detection techniques. In this study, reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay was devised to rapidly and specifically detect BCMV. Three pairs of specific primers were designed targeting the BCMV genome. To determine the ideal temperature, reactions were carried out across a temperature range of 45 °C to 70 °C, with intervals of 5 °C. The optimal temperature for the assay was determined to be 60 °C with a 30-min incubation period. Comparison between the RT-LAMP and conventional reverse transcription polymerase chain reaction (RT-PCR) revealed that former can detect the BCMV upto 10 and was one hundred times more sensitive than later. It was also determined that RT-LAMP was specific only in detecting BCMV, with no cross-reactivity with other closely related non-target viruses [potato virus Y (PVY), bean common mosaic necrosis virus (BCMNV), clover yellow vein virus (ClYVV) and soybean mosaic virus (SMV)]. After incubating the reactions at constant temperature of (60 °C/30 min), a characteristic ladder like banding pattern was observed on agarose gel for positive samples. Colorimetric tests (SYBR Green I) were also performed to reduce the requirement of laboratory equipment for visualizing RT-LAMP results. The results developed by SYBR Green I were comparable to that of agarose gel and can be visualized with naked eye. The developed RT-LAMP assay enables rapid detection of BCMV at 60 °C within a time period of 30-min.
菜豆普通花叶病毒（BCMV）是对豆科作物危害最严重的病毒之一。在绿豆中，BCMV是一种新出现的病毒，给作物造成巨大损失，从而降低了作物的产量和收益。作为一种种传和蚜虫传播的病毒，利用快速、特异且灵敏的检测技术来减少其传播并防止其转移到新的地理位置非常重要。在本研究中，设计了逆转录环介导等温扩增（RT-LAMP）检测方法来快速、特异检测BCMV。针对BCMV基因组设计了三对特异性引物。为确定理想温度，在45℃至70℃的温度范围内以5℃间隔进行反应。该检测方法的最佳温度确定为60℃，孵育时间为30分钟。RT-LAMP与传统逆转录聚合酶链反应（RT-PCR）的比较表明，前者能检测低至10的BCMV，比后者灵敏100倍。还确定RT-LAMP仅对检测BCMV具有特异性，与其他密切相关的非靶标病毒[马铃薯Y病毒（PVY）、菜豆普通花叶坏死病毒（BCMNV）、三叶草黄脉病毒（ClYVV）和大豆花叶病毒（SMV）]无交叉反应。在60℃恒温孵育反应30分钟后，阳性样品在琼脂糖凝胶上呈现出特征性的梯状条带模式。还进行了比色试验（SYBR Green I）以减少可视化RT-LAMP结果所需的实验室设备。SYBR Green I产生的结果与琼脂糖凝胶的结果相当，且可用肉眼观察。所开发的RT-LAMP检测方法能够在60℃下30分钟内快速检测BCMV。
