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 coinfecting viruses, affecting the growth, yield, and quality of kiwifruit. Therefore, a rapid and sensitive detection method is crucial for diagnosing and developing effective AcCRaV management strategies. 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 and 40°C within 10 to 30 min, and the amplification results can be read directly on the LFD within 5 min. The detection limit of the one-step RT-RPA-LFD assay was 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 accurate diagnosis of AcCRaV-infected kiwifruit plants in the field. To our knowledge, this is the first study applying the one-step RT-RPA-LFD assay to detect a kiwifruit virus.