Rapid and visual detection of Actinidia chlorotic ringspot-associated virus using one-step reverse transcription-recombinase polymerase amplification combined with lateral flow dipstick assay

Abstract

Actinidia chlorotic ringspot-associated virus (AcCRaV) occurs widely in major kiwifruit producing areas of China and is often accompanied by co-infecting viruses, affecting the growth, yield and quality of kiwifruit. Therefore, a rapid and sensitive detection method is crucial for the diagnosis, control as well as eradication of AcCRaV. In this study, a one-step reverse transcription-recombinase polymerase amplification combined with a lateral flow dipstick (RT-RPA-LFD) assay was developed for rapid detection of AcCRaV. Specific primers and a probe were designed based on the conserved region of the coat protein gene sequence of AcCRaV. The one-step RT-RPA reaction can be performed at 35 °C and 40 °C within 10 to 30 min, and the amplification results can be read directly on the LFD within 5 min. The detection limits of the one-step RT-RPA-LFD assay were 10-8 ng (about 20 viral copies), which was equal with one-step RT-qPCR and 100 times more sensitive than one-step RT-PCR. Moreover, the one-step RT-RPA-LFD assay was successfully applied to detect AcCRaV from crude extracts, and the entire detection process can be completed within 40 min. These results indicate that the RT-RPA-LFD assay is a simple, rapid and sensitive strategy that can be used for rapid diagnosis of AcCRaV-infected kiwifruit plants in the field. To our knowledge, this is the first study applying one-step RT-RPA-LFD assay to detect kiwifruit virus.