Development of a reverse transcription loop‐mediated isothermal amplification assay for rapid detection of strawberry crinkle virus

Abstract

AbstractStrawberry crinkle virus (SCV) has always been one of the major viruses affecting the strawberry production in China. Developing a quick and accurate method of detection is a crucial step for controlling the spread of SCV. In this study, a reverse transcription loop‐mediated isothermal amplification (RT‐LAMP) assay was developed for the detection of SCV. RNA was extracted from SCV‐infected strawberry leaves using a commercial extraction kit. According to the sequence of 1553 L protein (RdRp), four primers were developed for RT‐LAMP and a one‐step reaction was carried out at 65℃ for 30 min. Conventional reverse transcription Polymerase Chain Reaction (RT‐PCR) was performed as a comparison. The amplified RT‐LAMP products were separated on agarose gel by running electrophoresis and could be detected by visual inspection using Green‐DNA‐staining dye too. The detection sensitivity of the RT‐LAMP assay was 1000 times higher than that of the conventional RT‐PCR method, and no cross reaction was found with other strawberry viruses. Field samples with the positive rate of about 60% were collected to demonstrate the applicability of the RT‐LAMP for field detection and its high consistency with the traditional RT‐PCR method. The LAMP method described in this report is a sensitive, simple, economy and reliable assay for the rapid detection of SCV infection in strawberry plants.