220.127.116.11. Screening hive debris for SHB
Since adult SHB show cryptic behaviour, they are notoriously difficult to spot in hives. Moreover, beetles are highly migratory (Spiewok et al., 2008; Neumann et al., 2012) and may have left the hive prior to inspection. We therefore recommend the following molecular method to screen imported hives for SHB (modified after Ward et al., 2007:
- Extract DNA from hive debris samples:
1.1. Place ~10 g samples of debris into grinding mill canisters (Kleco, Visalia, California, USA).
1.2. Add CTAB (Hexadecyltrimethylammoniumbromide, [Sigma]) lysis buffer (12% Sodium phosphate buffer pH 8.0, 2% CTAB, 1.5 M NaCl), (20 ml) containing 1% antifoam B (Sigma) to each canister.
- Seal canisters.
- Load onto Kleco grinding mill.
- Ground for 2 min at top speed.
- Pour the lysate from each canister into a 50 ml Falcon tube.
- Spin the tubes at 4,000 g for 5 min.
- Remove 2 ml of the cleared lysate.
- Place into a 2 ml micro-centrifuge tube.
- Spin for a further 3 min at 10,000 g.
- Transfer cleared lysate (1 ml) to a fresh 2 ml micro-centrifuge tube.
- Add 250 µl of lysis buffer B (Promega) and 750µl of precipitation buffer (Promega).
- Spin tubes at 10,000 g for 10 min.
- Transfer clarified extract (750 µl) to a fresh micro-centrifuge tube.
- Add 50 µl of kit MagneSilTM beads and 600 µl of isopropanol.
- Incubate tubes at RT for 10 min.
- Extract DNA from each sample using the robotic magnetic particle processor (Kingfisher mL, Thermo Labsystems) in conjunction with the Promega DNA purification system for food kit following the instructions of the supplier.