Construction of genetically engineered Streptococcus gordonii strains to provide control in QPCR assays for assessing microbiological quality of recreational water.

Aims: 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.

Methods And 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.

Significance And Impact Of The Study: 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.