Rapid Detection of Tomato chlorosis virus from Infected Plant and Whitefly by One-step Reverse Transcription Loop-mediated Isothermal Amplification

Tomato chlorosis virus (ToCV) is the causal agent of an emerging virus disease of tomatoes which causes significant economic losses worldwide. The rapid spread of ToCV is associated with the increasing whitefly population in many countries. In this study, a highly efficient and practical one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) was developed for the rapid detection of ToCV in infected plant or whitefly. This RT-LAMP assay can detect ToCV either in total RNA or crude RNA extracted from infected plants using a water bath within 1h. The presence of ToCV in RT-LAMP products could be evaluated as ladder-like bands in an agarose gel or visualized in-tube with inclusion of a SYBR Green I dye under UV or daylight. RT-LAMP was 100-1000 times more sensitive compared to conventional RT-PCR for the detection of ToCV. RT-LAMP amplification was specific to ToCV, and no false reactions were detected when other viruses were tested. The developed RT-LAMP assay was also able to detect ToCV from purified RNA extracted from its vector whitefly. This is the first report of the application of the RT-LAMP to detect ToCV. The RT-LAMP assay developed in this study provides a rapid, sensitive and practical approach to facilitate the surveillance and management of ToCV.