Construction of genetically engineered Streptococcus gordonii strains to provide control in QPCR assays for assessing microbiological quality of recreational water.
OBJECTIVE
Quantitative polymerase chain reaction (QPCR) methods for beach monitoring by estimating abundance of Enterococcus spp. in recreational waters use internal, positive controls which address only the amplification of target DNA. In this study two internal, positive controls were developed to control for both amplification and cell lysis in assays measuring abundance of vegetative Gram-positive bacteria.


RESULTS
Controls were constructed using Streptococcus gordonii DL-1, a naturally transformable, Gram-positive bacterium. Unique target sequences were provided by chromosomal insertion of a genetically modified, green fluorescent protein gene fragment. Results suggest that their use for control of lysis and amplification may be of significant value.


CONCLUSIONS
The use of these controls and the establishment of data quality objectives to determine the tolerable level of decision error should ensure that environmental decisions based on QPCR data are technically and scientifically sound.


CONCLUSIONS
QPCR measurements related to cell abundance may vary between samples as thick-walled Gram-positive bacteria are inherently difficult to lyse and substances present in recreational waters may inhibit amplification. As QPCR methods are considered for beach monitoring, it is essential to demonstrate that the data obtained accurately reflects the abundance of the bacterial indicator.
James
J B
JB
US Environmental Protection Agency, National Health and Environmental Effects Research Laboratory, Gulf Ecology Division, Gulf Breeze, FL 32561, USA. james.joe@epa.gov
Genthner
F J
FJ
D004269:DNA, Bacterial / Q000235:genetics; D004784:Environmental Monitoring / Q000379:methods*; D005618:Fresh Water / Q000382:microbiology*; D005818:Genetic Engineering / Q000379:methods; D005822:Genetic Vectors; D016133:Polymerase Chain Reaction / Q000379:methods*; D054773:Streptococcus gordonii / Q000235:genetics* / Q000302:isolation & purification
D004269:DNA, Bacterial