Extraction and detection of baculoviral DNA from lake water, detritus and forest litter.
OBJECTIVE
This paper describes a quick, reproducible, sensitive method for baculoviral DNA extraction, purification and detection from freshwater and forest litter environments.


RESULTS
The extraction protocol utilizes enzymatic and chemical lysis and physical disruption. To assess the efficiency of the extraction and purification protocol, PCR was used to detect a 530 bp DNA fragment from the genome of a genetically-modified baculovirus, Choristoneura fumiferana NPVegt-/lacZ+. The detection limit of PCR amplification was routinely about 4.1 x 102 occlusion bodies (OBs) 450 microl-1 lake water. Template DNA from the detritus and forest litter samples required 100-fold dilutions before use in PCR reactions. The detection limits for detritus and forest litter samples were routinely about 7.41 x 103 and 2.08 x 104 OBs 0.5 g-1 dry weight, respectively.


CONCLUSIONS
The DNA extraction and purification methodology is reproducible, sensitive and can be used in lieu of, or in conjunction with, insect bioassays.


CONCLUSIONS
The DNA extraction and purification protocol described in this paper will facilitate risk assessment and ecological studies of both wild-type and genetically-modified baculoviruses.
England
L S
LS
Department of Natural Resources, Canadian Forest Service, Great Lakes Forestry Centre, Sault Ste Marie, Ontario, Canada.
Trevors
J T
JT
Holmes
S B
SB
D016367:Baculoviridae / Q000235:genetics / Q000302:isolation & purification*; D004279:DNA, Viral / Q000032:analysis; D004783:Environmental Microbiology; D016133:Polymerase Chain Reaction / Q000379:methods; D015203:Reproducibility of Results; D014871:Water Microbiology
D004279:DNA, Viral