A refined method for the detection of baculovirus occlusion bodies in forest terrestrial and aquatic habitats

A sensitive and efficient method was developed for the detection of genetically modified and wild-type baculovirus occlusion bodies (013) in forest terrestrial and aquatic habitats. The protocol facilitates the analysis of a large number of samples collected and frozen to maintain viral integrity. Lyophilization was used to standardize the size of both field-collected soil samples and test substrates inoculated with OBs for the determination of minimum detection threshold. To simulate natural conditions, terrestrial test substrates were inoculated at a standardized moisture content determined using a soil pressure plate apparatus. 013s, extracted from lyophilized test substrates by washing, sieving and centrifugation, were subjected to alkaline lysis and viral DNA isolated using a purchased DNA purification kit. PCR amplified DNA was visualized using agarose gel electrophoresis. Minimum detection thresholds in terrestrial substrates were 10(3), 10(2), 10(2) and 10(1) OBs from 0.5g of lyophilized L, F-H and mineral soil horizons, and 1.0ml of leachate, respectively. Detection thresholds in aquatic substrates were 10(0) and 10(3) OBs from 1.0ml of pond water and 1.0g of bottom sediment, respectively. (C) 2002 Society of Chemical Industry.