Rapid detection of apple stem grooving virus from reverse transcription recombinase polymerase amplification with a lateral flow dipstick (RT-RPA-LFD)

Abstract

Apple stem grooving virus (ASGV) is one of the most widespread and asymptomatic main viruses, that restricts the production of apples worldwide. Establishment of rapid, simple, and effective early detection methods of apple virus is important. In this study, we established and optimized a one-step reverse transcription - recombinase polymerase amplification (RT-RPA) method, using the target-specific primers of ASGV coat protein gene sequence, and M-MLV reverse transcriptase. This method could be completed within 30 min at 40 °C, followed by a visual detection of the results within 5 min by using lateral flow dipstick (LFD). The specificity results showed that only samples infected with ASGV showed a test line, while no test line appeared in the ASGV-negative samples. In addition, when crude extract of leaves was used, the whole detection could be completed within 1 h, which was shortened by 4 to 6 times compared with the RT-PCR method. The detection made on more field samples showed that the RT-RPA-LFD method is of high stability and reliability for ASGV diagnosis, with a great potential in the rapid on-site detection of plant viruses.

Keywords

• apple stem grooving virus
• reverse transcription - recombinase polymerase amplification
• lateral flow dipstick
• virus detection