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INTERNAL AMPLIFICATION CONTROL FOR USE IN QUANTITATIVE POLYMERASE CHAIN REACTION FECAL INDICATOR BACTERIA ASSAYS
Citation:
SIEFRING, S., E. ATIKOVIC, R. A. HAUGLAND, M. SIVAGANESAN, AND O. C. SHANKS. INTERNAL AMPLIFICATION CONTROL FOR USE IN QUANTITATIVE POLYMERASE CHAIN REACTION FECAL INDICATOR BACTERIA ASSAYS. Presented at OHIO BRANCH OF THE AMERICAN SOCIETY FOR MICROBIOLOGY, Canton, OH, April 13 - 14, 2007.
Description:
Quantitative polymerase chain reaction (QPCR) can be used as a rapid method for detecting fecal indicator bacteria. Because false negative results can be caused by PCR inhibitors that co-extract with the DNA samples, an internal amplification control (IAC) should be run with each sample. Currently available controls used in QPCR analyses for the fecal indicator bacterial groups Enterococcus and Bacteroidetes were designed primarily to determine variability in DNA yields from environmental samples and cannot be used to directly demonstrate PCR inhibition. Therefore, a competitive IAC plasmid DNA was constructed to detect the presence of PCR inhibitors in QPCR assays for both Enterococcus and Bacteroidetes rRNA gene targets. The IAC was designed to contain a single site for hybridization with a unique probe sequence that is flanked by multiple primer-hybridizing sites that corresponded to the same primers used in the Enterococcus, Bacteroidetes and several additional QPCR assays. The IAC construct was prepared by overlap extension PCR, inserted into the pCR4®TOPO plasmid vector (Invitrogen) and cloned. Gel electrophoresis, QPCR and sequencing analyses were performed to confirm the presence of the correct IAC sequences in the plasmid. Slope and intercept values of standard curves generated from genomic DNA in simplex analyses were not significantly different (p > 0.05) from the values generated during multiplex analyses with a fixed number of 25 IAC plasmid copies. Ranges of genomic DNA concentrations that did not significantly affect the IAC results under the same conditions were also established. Multiplex analyses with the IAC were used in a study of the relative levels of Enterococcus and Bacteroidetes DNA in fecal samples from cattle. In these analyses the Enterococcus IAC assay results showed highly consistent cycle threshold values (mean = 34.15, std. deviation = 0.69, N = 159) where only three results failed to occur within the 95% confidence interval established from analyses of control samples with IAC plasmid but no fecal extracts present. Greater variability in the Bacteroidetes IAC assay results was consistent with the relatively high levels of genomic DNA from these organisms in the samples. These studies indicate that the IAC plasmid DNA performs well as an inhibition control and also may be useful as an alternative to genomic DNA standards for quantifying fecal bacteria target DNA sequences.