Characterization and Establishment of a Recombinase Polymerase Amplification Assay for Rapid Detection of Tomato Spotted Wilt Virus in Pepper

Abstract Tomato spotted wilt virus (TSWV) is one of the most economically destructive and scientifically challenging plant viruses, which has seriously affected the production of commercial crops. At present, there is no effective strategy to control this virus. Therefore, there is an urgent need for a rapid and simple method to detect TSWV, which is of great significance to prevent its spread. In this study, an isolate of TSWV (TSWV-LNTL) infecting pepper from Liaoning Province of northeast China was obtained. A phylogenetic tree based on neighbor-joining using coat protein (CP) gene was established. A rapid method for detecting TSWV by recombinase polymerase amplification (RPA) was established. The phylogenetic tree based on the nucleotide sequences of coat protein (CP) genes of different TSWV isolates showed that the genetic relationship of TSWV-LNTL was most closely related to that of TSWV-LX-Lettuce-12 (Yunnan) and TSWV-TSHL (Shandong) isolates in China. It can be finished at 39 °C for 20 min and then purified by heating at 65 °C for 10 min. The RPA primers were highly specific and no cross-reactivity was detected with other selected viruses infecting pepper. The results of sensitivity test revealed that the detection limit of RPA is 1.0 × 103 copies/μL, which was tenfold lower than that of PCR method. In addition, the RPA method was successfully applied to detect TSWV in field samples. These results reported the occurrence of TSWV on crop in Liaoning Province of northeast China and demonstrated that the established RPA assay provided an effective molecular diagnostic tool for the accurate and rapid detection of TSWV to prevent its spread.