Development and validation of LAMP and real-time LAMP assays for rapid detection of coccinia mosaic Virudhunagar virus in bitter gourd (Momordica charantia).

Abstract

Mosaic disease, caused by whitefly-transmitted begomoviruses, significantly threatens bitter gourd cultivation in India. This study identified and characterized the complete genome of coccinia mosaic Virudhunagar virus (CoMViV) from symptomatic bitter gourd samples collected in Oddanchatram block of Dindigul district, Tamil Nadu, using rolling circle amplification and sequencing. A loop-mediated isothermal amplification (LAMP) protocol was optimized by targeting regions of the CoMViV AV1 and AV2 genes. The LAMP assay demonstrated exceptional sensitivity, detecting as little as 10 fg of viral DNA, a 100-fold improvement compared to PCR. Specificity tests confirmed that the LAMP assay exclusively detected CoMViV in bitter gourd, with no cross-reactivity with other begomoviruses. Real-time LAMP assay produced a distinct annealing peak at 86.5 ± 0.5 °C. Furthermore, viral quantification revealed a maximum viral titer of 3.2 × 10⁹ copies per µl in infected leaf samples. While both LAMP and PCR assay successfully detected CoMViV in infected tissues, the LAMP assay offers advantages including simplicity, rapid, and sensitivity. This optimized LAMP assay provides a valuable tool for differentiating CoMViV from other begomoviruses infecting bitter gourd crop, offering an improvement over existing PCR assay. To the best of scientific knowledge, we, for the first time, demonstrate the LAMP assay for the detection and quantification of CoMViV in bitter gourd plants.

Supplementary information: The online version contains supplementary material available at 10.1007/s13205-025-04484-2.