Evaluation of PCR amplification-based detection of heat-killed Escherichia coli and cell-free DNA in shellfish. Molecular Biology Today 3

Presence of E. coli is a useful indicator of fecal contamination in estuarine shellfish. Polymerase chain reaction using primers for uidA were used to detect DNA released from dead E. coli cells and from heat-killed cells in seeded oyster tissue homogenate. Two different methodologies were adopted for DNA extraction from the seeded oyster homogenate: Chelex ™ 100 method and a modified cell-lysis method. Results showed PCR amplification of DNA purified by the modified cell-lysis method was able to eliminate detection of cell-free DNA or heat-killed non-viable cells at various concentrations tested as opposed to the Chelex ™ 100 method. DNA-PCR detection was positive for uidA using the Chelex ™ 100 DNA extraction method on oyster samples with high concentrations (6-100µg) of cell-free DNA or heat-killed cells (1 X 106 cells/ml). This suggests that a difference in DNA extraction techniques can eliminate false-positive results by PCR, thus making it a reliable and rapid diagnostic tool to differentiate viable and non-viable cells in shellfish samples. It would greatly enhance the microbiological safety of shellfish and the quality of risk-assessment involved in the detection of viable microbial pathogens in seafood samples