2.6. PCR Diagnosis of Sentinel Eggs

The DNA from each egg was extracted using the Chelex protocol described above and screened with the species-specific T. japonicus PCR assay. In order to avoid false negatives and increase the detection of small quantities of DNA, all egg extractions were subjected to reamplification using 1 µL of PCR product from the first round of PCR as a template. This was repeated so each egg was screened using reamplification at least twice. Negative controls were included to check for cross-contamination. Reamplification bands were visualized on 2% SeaKem agarose (Lonza, Rockland, ME, USA) pre-stained with GelRed nucleic acid gel stain (1X; Biotium, Hayward, CA, USA). Reamplification could be particularly important to detect trace amounts of parasitoid DNA in empty eggs following parasitoid emergence (code B). Other studies have shown that empty parasitized eggs 7 d post-emergence have about a 50% amplification rate for parasitoid DNA [29]. This is congruent with our results for eggs of H. halys and P. maculiventris that were about 6 weeks post-emergence (55% PCR positive for T. japonicus). For C. hilaris, none of the empty parasitized eggs were positive for T. japonicus. However, our sample size was very low (n = 3). The relatively poor detection of parasitoid DNA in eggs from which the adult has eclosed is not unexpected, given that most of the DNA was removed with the adult.