(a) Thrip samples collection
Thrip adult and larvae samples were collected on imported plants by the Bureau of Animal and Plant Health Inspection and Quarantine at Tao-Yuan International Airport in Taiwan. Samples were stored in screw thread vials (7 ml) containing 70 per cent ethanol at 4°C for use.
(b) Total genomic DNA extraction
Total genomic DNA was extracted from whole thrip adults and larvae using the EasyPure genomic DNA spin kit (Bioman), according to the manufacturer's instructions. Briefly, samples were lysed with 200 µl ml−1 of proteinase K, and then incubated in 200 µl of buffer. Genomic DNA was precipitated in a column with 200 µl of ethanol, followed by two washing steps. Finally, the genomic DNA was eluted in 30 µl of diethyl pyrocarbonate (DEPC) treated water.
(c) Primers and probes designed for Taqman real-time quantitative PCR (qPCR) detection system
The ribosomal RNA gene sequences of the trips were downloaded from GenBank to design primers. The thrips included F. occidentalis, Frankliniella intonsa and Frankliniella schultzei (GenBank accession number GQ343254, GQ343258, GQ343259). We designed two primer and probe sets targeted on 5′ and 3′ regions of the ribosomal RNA gene specifically for F. occidentalis ().
Primers and probes used in this study.
(d) Taqman real-time quantitative PCR (qPCR)
Taqman real-time quantitative PCR was performed with LightCycler® 480 machine and LightCycler® 480 Probes Master kit (Roche). Reactions were performed in a final total volume of 10 µl containing 1× LightCycler 480 Probes Master, 0.5 µM forward primer, 0.5 µM reverse primer, 0.2 µM Taqman probes and 2.5 µl of the DNA template. Amplification conditions were as follows: denaturation at 95°C for 10 min, 50 cycles of amplification (95°C for 10 s, 60°C for 30 s) and cooling at 40°C for 10 s. All samples were tested by qPCR for both 5′ and 3′ regions of the ribosomal RNA gene for F. occidentalis.
(e) Specificity of detection system
Seven different thrip species, including F. schultzei, Scirtothrips dorsalis, Thrips tabaci, Caliothrips fasciapennis, Tubulifera, F. intonsa and F. occidentalis were used to evaluate the specificity of the primer/probe sets.
(f) Standard curve of detection system
Taqman qPCR standard curves were established on 10-fold serial dilutions of the thrip genomic DNA from 10 ng µl−1 to 0.1pg µl−1 to estimate in tetraplicate the system's linear range, efficiency and detection limitation for both 5′ and 3′ regions.