Overcoming the Inhibitor Problem in PCR Detection of Salmonella in Recreational Waters: A Volume Based Approach

Fecal contamination of environmental or recreational waters is a world wide health concern. Methods currently endorsed by the U.S. EPA for monitoring fecal contamination are based on plate counts of fecal surrogates such as E. coli or Enterococci spp. Due to the sensitivity and specificity of PCR, there is growing interest in using PCR-based methods to detect and quantitate pathogens directly. However, a major impediment to more widespread use of PCR-based methods is the presence of inhibitors that frequently contaminate DNA isolated from environmental sources, especially water rich in organic matter. The major objectives of the present project are to ascertain the extent of this inhibitor problem and to develop alternative approaches. Using Salmonella as a model, real-time PCR and an Amplification Control (AC), we demonstrate that PCR inhibition occurs to varying degrees with all environmental water samples. Inhibition can be ameliorated by using less template DNA but the level of dilution required to permit amplification of the AC is often too severe to permit detection of the pathogen. Our results show that because the severity of the inhibitor problem is stochastic and there is not a reliable method to correct for the effect of the inhibitor, it is not feasible to convert real-time PCR data to bacterial counts. Instead, we propose a volume based approach to quantitate pathogens in recreational waters. The method is based on realtime PCR detection of bacteria, Salmonella in this case, in a specific volume of water. It includes a short enrichment step in a non-selective medium prior to DNA isolation and PCR. The enrichment step increases the amount of template DNA isolated without a concomitant increase in inhibitor level and solves the inhibitor problem because the ratio of inhibitors to template DNA used in PCR is greatly reduced. The detection limit of the method was 10 CFU/dL using a 6 hr enrichment period. Because the risk of exposure to a particular waterborne pathogen is directly related to its concentration, we believe this volume based approach provides a useful method to assess exposure risk in the future