Make It “Tough”: qPCR Master Mix Comparison
Poster Presentation
Prepared by M. Citriglia, N. Schafer
Northeast Ohio Regional Sewer District, 4747 East 49th Street, Cuyahoga Height, OH, 44125, United States
Contact Information: CitrigliaM@neorsd.org; 216-641-6000
ABSTRACT
Within the 2012 Recreational Water Quality Criteria guidelines, the EPA provides information for states who want to adopt Water Quality Standards based on rapid methods that EPA has developed and validated using quantitative polymerase chain reaction (qPCR). Currently, the EPA has validated Method 1609 and 1611 for the quantification of Enterococcus in environmental waters using qPCR. The NEORSD laboratory has collected samples from two Northeast Ohio Beaches and analyzed them by EPA method 1609 and a qPCR assay for E. coli. The objectives of this study was to identify a master mix that (1) could be used for both assays, (2) reduce inhibition, (3) is similar to the Environmental Master Mix (EMM) recommended by the EPA, (4) utilizes an Internal Amplification Control (IAC) allowing for analysis on the same platform, and (5) contains a purified DNA polymerase that is validated free of extraneous E. coli DNA. A total of 241 samples were analyzed for both organisms using two different chemistries, EMM as stated in method 1609 and Tough Mix. The data for each organism and master mix combination was compared for inhibition, method QC, level of quantification and difference between assay absolute and relative quantitation. The accuracy, sensitivity, and specificity were calculated as compared to the conventional method. In conclusion, we have determined that the Tough Mix yielded comparable data to the EPA recommended mix with respect to the accuracy and reproducibility, but the level of inhibition and instance of positive QC data decreased with the Tough Mix.
Poster Presentation
Prepared by M. Citriglia, N. Schafer
Northeast Ohio Regional Sewer District, 4747 East 49th Street, Cuyahoga Height, OH, 44125, United States
Contact Information: CitrigliaM@neorsd.org; 216-641-6000
ABSTRACT
Within the 2012 Recreational Water Quality Criteria guidelines, the EPA provides information for states who want to adopt Water Quality Standards based on rapid methods that EPA has developed and validated using quantitative polymerase chain reaction (qPCR). Currently, the EPA has validated Method 1609 and 1611 for the quantification of Enterococcus in environmental waters using qPCR. The NEORSD laboratory has collected samples from two Northeast Ohio Beaches and analyzed them by EPA method 1609 and a qPCR assay for E. coli. The objectives of this study was to identify a master mix that (1) could be used for both assays, (2) reduce inhibition, (3) is similar to the Environmental Master Mix (EMM) recommended by the EPA, (4) utilizes an Internal Amplification Control (IAC) allowing for analysis on the same platform, and (5) contains a purified DNA polymerase that is validated free of extraneous E. coli DNA. A total of 241 samples were analyzed for both organisms using two different chemistries, EMM as stated in method 1609 and Tough Mix. The data for each organism and master mix combination was compared for inhibition, method QC, level of quantification and difference between assay absolute and relative quantitation. The accuracy, sensitivity, and specificity were calculated as compared to the conventional method. In conclusion, we have determined that the Tough Mix yielded comparable data to the EPA recommended mix with respect to the accuracy and reproducibility, but the level of inhibition and instance of positive QC data decreased with the Tough Mix.
