Rapid detection of apple latent spherical virus infecting Angelica sinensis by gold immunochromatography reverse transcription loop-mediated isothermal amplification assay

Abstract

Apple latent spherical virus (ALSV; genus: Cheravirus, family: Secoviridae) is capable of infecting Angelica sinensis under natural conditions and causing symptoms of greenish discoloration and mottling, affecting plant growth and causing a reduction in root quality and yield. Virus detection of ALSV is an urgent need for the continued development of angelica industry. Consequently, it is necessary to develop simple, sensitive, and reliable ALSV detection methods. We describe the preparation of polyclonal antibodies with high titer and high specificity using recombinant ALSV coat protein (CP) as the antigen. Using these antibodies, we established and optimized a rapid detection system using colloidal gold immunochromatography assay (GICA) to detect ALSV. A real-time reverse transcription loop-mediated isothermal amplification (RT-LAMP) system with a minimum detection line of 2.72 × 103copys/μl was also developed to detect ALSV. This assay was further enhanced using a combination GICA-RT-LAMP assay. This GICA assay successfully detected ALSV in infected plants without cross reactivity recorded from three other plant viruses. The sensitivity of GICA was the same as that of GICA-RT-PCR, whereas the sensitivity of GICA-RT-LAMP was at least 10-fold higher than that of GICA and GICA-RT-PCR. These methods can detect and identify ALSV under field conditions and accurately measure virus concentration in the laboratory.