Development of reverse transcription loop-mediated isothermal amplification (RT-LAMP) for rapid detection of viruses infecting patchouli (Pogostemon cablin).

Abstract

Patchouli (Pogostemon cablin), a highly valued medicinal plant, suffers significant economic losses following infection with Broad bean wilt virus 2 (BBWV-2) 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 detection of BBWV-2 and PStV. The oligo primers were designed to target the coat protein genes of PStV and BBWV-2. The reaction conditions, such as temperature and time duration, were optimized to 65°C for 60min. The LAMP amplicons positive for PStV and BBWV-2 revealed characteristic ladder-type bands following agarose gel electrophoresis. Further, a colorimetric assay using a metal ion-based indicator (Hydroxy-naphthol blue, HNB) was conducted to visualize the amplified products with the naked eye, thus facilitating accessibility to field practices. The assay developed in this study was found to be virus specific, and was 100 times more sensitive than RT-PCR. Thus, the RT-LAMP assay established in this study is quick, reliable, and cost-effective for the accurate identification of BBWV-2 and PStV. It will facilitate the screening of patchouli planting materials. Further, it may reduce the risk of virus spread and could be helpful in phytosanitary programs.