Rapid visual detection of Japanese hornwort mosaic virus infecting Angelica sinensis by reverse transcription loop‐mediated isothermal amplification

Abstract

AbstractJapanese hornwort mosaic virus (JHMV; genus Potyvirus, family Potyviridae) 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 Mg2+ at 60°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 (κ) 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 in A. sinensis in China.