Development of Reverse Transcription Loop Mediated Isothermal Amplification (RT-LAMP) for rapid detection of viruses infecting patchouli (Pogostemon cablin)

Abstract

Abstract Patchouli (Pogostemon cablin) a highly valued medicinal plant, suffers significant economic losses following infection with Broad bean wilt virus 2 (BBWV2) and Peanut stripe virus (PStV). In this study, a field-based isothermal technique called reverse transcription loop-mediated isothermal amplification (RT-LAMP) was established for an early and specific identification of BBWV2 and PStV. The oligo primers for RT-LAMP assay were designed from the conserved domains of the coat protein genes of PStV and BBWV2. The reaction temperature and time duration were optimised to 65 0C for 60 min, followed by incubation at 85 0C for 10 min in a water bath. The LAMP amplicons positive for PStV and BBWV2 revealed characteristic ladder-type bands when analysed through gel electrophoresis. Further, a colorimetric assay using a metal ion-based indicator (Hydroxy-napthol Blue, HNB) was conducted to visualize the amplified products with the naked eye, thus facilitating accessibility to field practices. The assays developed in this study were virus-specific as no cross-amplification occurred with other viruses, and were 100 times more sensitive than RT-PCR. Thus, the RT-LAMP assays established in this study are quick, reliable, and cost-effective for the accurate identification of BBWV2 and PStV. It will facilitate screening of patchouli planting materials. Thus, may reduce the risk of virus spread and could be helpful in phytosanitary programs.