Development of a novel, rapid, and accurate recombinase polymerase amplification assay for the identification of the glassy-winged sharpshooter, Homalodisca vitripennis (Hemiptera: Cicadellidae).

Abstract

The glassy-winged sharpshooter (GWSS; Homalodisca vitripennis) is a vector for the bacterium Xylella fastidiosa, the causative agent of Pierce's disease. Here, we have developed and optimized an isothermal recombinase polymerase amplification assay (RPA) to detect GWSS based on the Internal Transcribed Spacer 2 (ITS2) region. The assay is 100% specific for GWSS (five target and 12 non-target specimens tested). It can detect specimens collected from diverse geographic regions, and no cross-reactions were observed with non-target cicadellids. The assay can effectively detect the presence of GWSS DNA as low as 3 pg. The RPA assay is tenfold less sensitive than real-time PCR assay but three times faster (less than 15 min for RPA vs. 45 min for real-time PCR). Another major advantage of this newly developed RPA assay is that it can be deployed very easily at the sampling site as it does not require any sophisticated instrumentation.