Wednesday, December 13, 2006

A system for rapid molecular identification of immature Anastrepha (Diptera: Tephritidae) intercepted at ports of entry

Don C. Vacek,, Heather A. Quintanilla,, and Lisa A. Ledezma, USDA-APHIS-PPQ-CPHST, Pest Detection, Diagnostic, and Management Laboratory, Moore Air Base Bldg.6414, 22675 N. Moorefield Rd, Edinburg, TX

Standard block PCR protocols with Anastrepha species specific primer pairs have been adapted to real time PCR with SYBR Green and TaqMan chemistry. Species specific target sequences of 50 to 600 base pairs have been successfully amplified with real time methodology. Diagnoses from real time plots of known species and intercepted immatures have been confirmed by electrophoresis of the products from each real time PCR reaction. Anastrepha ludens, A. obliqua, and A. serpentina can be identified with real time PCR with SYBR Green in 2 hours and confirmed with electrophoresis. This is a significant improvement over fingerprinting methods (2 days). A molecular diagnostic with primer-probe, TaqMan chemistry has been developed for Anastrepha obliqua and has been confirmed with 10+ individuals from populations of six Anastrepha species of economic inportance.

Species 1: Diptera Tephritidae Anastrepha ludens (Mexican fruit fly)
Species 2: Diptera Tephritidae Anastrepha obliqua
Species 3: Diptera Tephritidae Anastrepha serpentina