Persistence of extracellular baculoviral DNA in aquatic microcosms: extraction, purification, and amplification by the polymerase chain reaction (PCR)

Genetically-modified baculoviruses have potential uses as bio-pesticides in forestry. However, the baculoviral occlusion bodies (OBs) may release genetically-modified DNA into the forest environment. In this research, outdoor aquatic microcosms, spiked with 673 μg of genomic DNA (4.4×1012 target copies) from the genetically modified baculovirus Choristoneura fumiferana MNPVegt−/lacZ+, were exposed to natural summer conditions. A 530 bp DNA fragment from the genome of CfMNPVegt−/lacZ+ was detected in field microcosm water samples for about 24 h. The introduced DNA may have persisted for a longer time, but was below the detection limit of the PCR analysis (13.5 pg DNA or 8.9×104 target copies ml−1 water). The detection limit of PCR was determined by spiking water samples with a dilution series of CfMNPVegt−/lacZ+ genomic DNA, extracting and purifying the DNA, and then PCR analysis. This study provides some of the first information on the persistence and detection limits of this viral DNA under aquatic ecological conditions, and the methods that can be used to conduct such a molecular-based field study.