A sequence specific real-time PCR (TaqMan®) assay for Thrips palmi (Thysanoptera: Thripidae); Its use and advantages as a molecular quarantine identification tool
Collins DW, Walsh K, Boonham N, Glover R, Barker I
Central Science Laboratory, Sand Hutton, York YO41 1LZ, United Kingdom.
Correspondence: dom.collins@csl.gov.uk
The polyphagous pest Thrips palmi Karny has become a species of major quarantine concern, often intercepted on plant material in international trade. The ability to rapidly identify the species is a critical factor that will help determine the success of any campaign to prevent its establishment in Europe. Confirmed morphological identification to species is limited to the adult thrips, yet quarantine diagnostic laboratories frequently have to make identifications with only larvae available, often with perishable commodities at stake. In this study, RAPD analysis was performed to identify 19 putative SCAR markers, which were screened by southern blot analysis; one marker was sequenced and a real-time PCR (TaqMan) assay developed (Walsh K et al. 2005. Journal of Applied Entomology 129: 272¨C279). The assay was screened against 21 thrips species including 10 other species of the genus Thrips and found to be specific to T. palmi. The speed, specificity, simplicity and robustness of the assay are all reasons why a real-time PCR format was selected for specific use as a quarantine entomology diagnostic tool in preference to other available molecular techniques. The use of a Smart Cycler II TD (Cepheid) offers the possibility of further reducing the total time of this assay to as little as 45 minutes, as well as the potential for on-site testing of samples at a suspected outbreak site or at a port-of-entry. The assay was designed to complement morphological identification of thrips, not to replace it. Numerous different species of thrips have been found in trade, both major pest species and some quite obscure ones. Morphological expertise will continue to be required to deal with the complexity and range of identification work required. Nevertheless, the development of molecular tools of ever increasing speed and scope offer tools for the future that will further increase the efficiency of quarantine diagnostic laboratories. To that end, the cytochrome oxidase I gene from those thrips species and populations used to produce this assay have been sequenced and analyzed as the first step towards the production of a microarray protocol with the ultimate aim of producing an assay that offers simultaneous screening of numerous thrips species in a user-friendly format.
