Development of thrips barcode database and multiplex real-time PCR assay for quarantine and agriculture pest species

Thrips (Order Thysanoptera) species are agriculturally important as plant sap sucking pests and vectors of several plant diseases. They are very small insects and commonly associated with imported commodities at New Zealand border in all life stages. Morphological identification of thrips is mainly performed on adults, but the available identification keys for immature stages do not include many species and are inadequate, thus DNA barcode was regularly used for thrips identification, here, we have generated DNA barcode data for over 29 thrips species from over 100 individuals.  At New Zealand border, Frankliniella occidentalis is the dominant species intercepted, followed by F. panamensis, Thrips palmi and T. tabaci and several other thrips species. Hence, we have also developed a multiplex real time PCR assay, targeting the four thrips species to facilitate the identification of quarantine interceptions with more accurate and faster diagnostic method for any developmental stages. The DNA barcode database further assists in thrip identification. The assay showed high specificity for all the four target species and could detect 10 copies/ µL of the target DNA. Linear responses and high correlation coefficients between the amount of DNA and Cq values for each species were also achieved. The method was tested on single egg, larva and adult and proved to be applicable for all life stages of the four species. This study has demonstrated the assay is a useful biosecurity tool for rapid and reliable identification of the target thrips species.