Rapid visual detection of Japanese hornwort mosaic virus infectingAngelica sinensisby reverse transcription loop-mediated isothermal amplification

Japanese hornwort mosaic virus (JHMV; genusPotyvirus, familyPotyviridae) is a widespread virus that infects angelica (Angelica sinensis[Oliv.] Diels), an important Chinese herbal medicine plant grown in Gansu, China. JHMV infection has contributed to the deterioration in angelica quality and a reduction in yield. Consequently, there is a need to develop a reliable, simple and rapid detection method to accurately identify JHMV infection and help limit its spread. We describe here, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) developed to detect the coat protein gene of JHMV. RT-LAMP amplification products were assessed through real-time fluorescence detection and by gel electrophoresis and SYBR Green I DNA staining for visual observation. This assay successfully detected JHMV in infected plants without cross reactivity recorded from six other plant viruses. Optimum LAMP reactions were conducted in betaine-free media with 6 mM Mg(2+)at 60 degrees C for 60 min for JHMV. The detection limit was 0.28 pg/ml using RT-LAMP for JHMV plasmids. This detection limit for the RT-LAMP assay was 100 times lower than that of the conventional RT-polymerase chain reaction (RT-PCR) assay. Our field survey of angelica crops for JHMV using RT-LAMP further demonstrated a higher sensitivity than RT-PCR, detecting 78% versus 72%. Agreement (kappa) between the results obtained from the two tests was 0.844. We found RT-LAMP is accurate and efficient in diagnosis and potentially improving JHMV disease management and forecasting inA. sinensisin China.

Automated summary

The study introduces a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method for detecting Japanese hornwort mosaic virus (JHMV) in angelica plants. This RT-LAMP assay showed higher sensitivity and lower detection limit compared to conventional RT-PCR, proving to be accurate and efficient for JHMV diagnosis. Field surveys using RT-LAMP demonstrated improved disease management and forecasting capabilities for JHMV in Angelica sinensis in China.