Reverse transcription recombinase polymerase amplification assay for rapid detection of the cucurbit chlorotic yellows virus

Abstract

The cucurbit chlorotic yellows virus (CCYV) causes severe economic losses in cucurbit plants. Although it has been widely known in various countries for several years, CCYV is rarely recognized due to the lack of rapid and effective detection methods in the early stage of the disease. Recombinase polymerase amplification (RPA) is a new, efficient, and simple technology for nucleic acid detection. In the present study, reverse transcription (RT)-RPA and quantitative RT-RPA were developed and utilized for fast detection of CCYV in field-collected melon samples. The analysis was performed under constant temperature conditions without the necessity for a thermal cycler in just 20 min. Moreover, the detection limit of RT-RPA for CCYV was determined at 10 pg. In the study, 58 field-collected samples were employed to evaluate the performance of the two assays. The positive rates were established at 72.4 % (42/58) and 75.9 % (44/58) by RT-RPA and qRT-RPA, respectively, and were consistent with the RT-PCR results. The successful application of RPA for the detection of CCYV in field-collected melon samples indicated its potential applicability. Thus, the developed RPA assays provide an alternative for fast, efficient, sensitive, and reliable detection of CCYV in diagnostic laboratories, which lack the precise instrumentation, and fields without appropriate equipment.