Development of a reverse transcription-loop mediated isothermal amplification assay for detection of wisteria vein mosaic virus

Wisteria is a woody, deciduous, ornamental, perennial climbing vine belonging to the pea family (Fabaceae), one of the largest families of flowering plants. Wisteria vein mosaic virus (WVMV) is one of the most destructive viruses of wisteria. The virus causes mild mosaic mottling, chlorotic spots, necrotic flecks, and distortion or twisting, as well as a reduced flowering ability, making them lose their ornamental value. To reduce the economic losses caused by WVMV, the establishment of efficient, rapid and accurate early diagnosis technology is the foundation for effective prevention and control of WVMV. In this study, a reverse transcription loop-mediated isothermal amplification (RT-LAMP) method was established for the detection of WVMV. The optimal reaction components were determined to be 0.5 mM dNTPs, 0.6 M betaine, 4 mM Mg2+, and the best ratio of the inner primers (FIP and BIP) to the outer primers (F3 and B3) was 4:1. The sensitivity of the RT-LAMP detection method is 100-fold higher than that of the conventional PCR, which reaches the same sensitivity of qPCR. The RT-LAMP specifically detected WVMV, and no cross-react with common virus was observed. To our knowledge, this is the first diagnostic assay based on RT-LAMP for a viral pathogen in wisteria. The developed RT-LAMP assay in this study was found to be robust, simple, specific, rapid and sensitive enough to have the potential to be used in early diagnosis of WVMV in the field.