Real-time detection of cucurbit chlorotic yellows virus by RT-LAMP

Cucurbit chlorotic yellows virus (CCYV) of the genus Crinivirus and family Closteroviridae, is an emerging infectious agent transmitted by whiteflies that mainly infects cucumber, melon and watermelon plants. Here, we developed a procedure based on reverse transcription (RT) followed by loop-mediated isothermal amplification (LAMP) to detect CCYV in real-time by using a fluorescent dye and incubating at 65 °C. This method detected CCYV in RNA extracts at about 10 min and in nylon membrane-filtered crude extracts between 55 and 95 min. The detection was sensitive, ten times higher than RT followed by polymerase chain reaction (PCR) and specifically detecting different isolates of CCYV without cross-reaction with other viruses of the same genus. This procedure enables the simultaneous analyses of multiple samples, allowing for rapid, sensitive and specific detection of CCYV. By using membrane-filtered crude extracts, RNA purification is unnecessary, so the whole process can be performed in the field with a portable heater and fluorometer, facilitating a rapid response for disease control.