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

被引:17
作者
England, LS
Pollok, J
Vincent, M
Kreutzweiser, D
Fick, W
Trevors, JT [1 ]
Holmes, SB
机构
[1] Univ Guelph, Ontario Agr Coll, Dept Environm Biol, Lab Microbial Technol, Guelph, ON N1G 2W1, Canada
[2] Canadian Forest Serv, Dept Nat Resurces Canada, Great Lakes Foresty Ctr, Sault Ste Marie, ON P6A 2E5, Canada
基金
加拿大自然科学与工程研究理事会; 加拿大创新基金会;
关键词
amplification; aquatic; detection; DNA; forest environment; persistence;
D O I
10.1016/j.mcp.2004.09.004
中图分类号
Q5 [生物化学];
学科分类号
071010 ; 081704 ;
摘要
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 mug Of ggenomic DNA (4.4 x 10(12) 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 x 10(4) 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 Linder aquatic ecological conditions, and the methods that can be used to conduct such a molecular-based field study. (C) 2004 Elsevier Ltd. All rights reserved.
引用
收藏
页码:75 / 80
页数:6
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