DIRECT PCR
The water sample is decimally diluted, and each dilution is used as a substrate for PCR.


E. coli O157:H7 cells (103) were added to 50 ul of either double distilled water or groundwater. Decimal dilutions were carried out to a theoretical concentration of 10^-2 cells and each tube was used in DPCR with the stx2 primer pair. The negative control without added template did not yield a PCR product (not shown). M, molecular weight markers.

A stx2 product (confirmed by sequencing) was formed in every tube with sufficient template. One cell was sufficient to obtain a product. No PCR product was formed in undiluted groundwater with 103 cells due to inhibition by materials present in the groundwater. This inhibition was relieved by dilution. The use of DPCR for detection of E. coli O157:H7 or other Shiga-toxin producing bacteria in water samples would require 20 cells/ml (200 cells/ml for those samples requiring 1:10 dilution).

Project Summary:
We have developed an improved approach to the detection and quantification of bacteria in water. Direct PCR (DPCR), is a highly sensitive method that is rapid and relatively simple. DPCR incorporates samples directly in the PCR reaction and obviates the need for recovery of cells from the sample or DNA extraction. Because DPCR minimizes steps in sample handling, it can readily be scaled up to process a large number of samples. We have applied DPCR to the detection of Escherichia coli O157:H7 and Vibrio cholerae. These are among the highest priority waterborne pathogens identified as biological threats by the CDC. Genes encoding virulence features of these pathogens were used as targets for primers.
 
E. coli O157:H7 was added to distilled water and to groundwater. Serial dilutions were used as templates in DPCR. One cell could be detected. V. cholerae was added to drinking water, serially diluted, and used as template in DPCR. Ten to 100 cells were required for detection. In addition, DPCR was used to detect Helicobacter pylori in a naturally contaminated drinking water sample. Drinking water from a municipality in the southeastern U.S. was concentrated 40-fold and subjected to DPCR. A product indicating H. pylori contamination was obtained. While this organism causes chronic rather than acute disease and would not be expected to be used as a bioweapon, the successful detection of H. pylori in drinking water suggests that DPCR is a useful method that should be widely applicable to the detection of pathogens in water.

As with other PCR-based methods, a positive reaction does not mean that a sample has live, infectious bacteria because dead cells or free DNA would also serve as a template. However, the ability of DPCR to detect dead cells or DNA could be advantageous for some purposes. For example, DPCR could be useful in tracing the source of an outbreak to identify a pathogen in a sample regardless of whether it still contains viable cells. In contrast to PCR-based methods, culture-based methods can result in false negatives due to the failure to detect viable nonculturable cells that may be infectious.