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DEVELOPMENT OF DATA QUALITY OBJECTIVES AND USE OF TWO VARIATIONS OF GENETICALLY-MODIFIED STREPTOCOCCUS GORDONIL AS LYSIS CONTROLS IN A QPCR ASSAY FOR ASSESSING SANITARY QUALITY OF WATER
Citation:
JAMES, J. B. AND F. J. GENTHNER. DEVELOPMENT OF DATA QUALITY OBJECTIVES AND USE OF TWO VARIATIONS OF GENETICALLY-MODIFIED STREPTOCOCCUS GORDONIL AS LYSIS CONTROLS IN A QPCR ASSAY FOR ASSESSING SANITARY QUALITY OF WATER. Presented at 107th ASM General Meeting, Toronto, ON, CANADA, May 21 - 26, 2007.
Description:
Joseph B. James and Fred J. Genthner
United States Environmental Protection Agency, Gulf Breeze, FL
Background: Methods using rapid cycle, real-time, quantitative (QPCR) are being developed for detecting and quantifying Enterococcus spp. as well as other aquatic bacterial indicators. With all molecular methods, employment of good controls assures the validity of the assay. It is essential to demonstrate that differences in fluorescence are attributable to bacterial abundance, not to differences in lysis or amplification efficiencies. Two lysis and amplification bacterial controls were developed. In each, an oligonucleotide was inserted into the chromosome of Streptococcus gordonii (DL-1), a naturally transformable, Gram-positive bacterium found in dental caries. Data quality objectives were developed for use of these controls. Methods: Two controls were developed. For the first, a 97 bp fragment, based on a 176 bp stretch of the green fluorescent protein (GFP) gene from great star coral, provided a unique target sequence. For the second, the same GFP fragment was used, with modifications to include 16S rRNA gene-based, enterococcus-specific primer sequences resulting in an amplicon of the same length and GC content (145bp, 51% GC content) as a 16S ribosomal DNA-based, enterococcus-specific, amplicon designed for detection and quantification. The second construct was designed specifically with Enterococcus spp. detection in mind, while the first was constructed as a control for general use. QPCR was used to detect the constructs in known numbers of S. gordonii cells, allowing for evaluation of lysis success. Conclusion: S. gordonii strains, possessing unique target sequences integrated within the chromosome, provide lysis controls for QPCR analyses. One construct also serves as an inhibition control for a specific Enterococcus assay.
Topic: Q16 Methods in Environmental Microbiology
Keywords: Enterococci, lysis control
ASM Membership: J. B. James
Contact Information: Joseph B. James, US EPA Gulf Ecology Division, 1 Sabine Island Drive, Gulf Breeze, FL 32561. (office) 850.934.2457, (fax) 850.934.9201 james.joe@epa.gov