Quantitative detection of Plesiomonas shigelloides in clam and oyster tissue by PCR

A quantitative assay for Plesiomonas shigelloides in clams and oysters based on the conventional polymerase chain reaction was developed. The assay involved the treatment of homogenized tissue samples with 4.0% formaldehyde that presumably denatured DNases and proteases present in the tissue which would otherwise inactivate the PCR reaction. The level of detection of P. shigelloides in clam tissue without enrichment was 200 CFU/g. The addition of 0.1% bovine serum albumin (BSA) to PCR reactions or the DNA purification system reduced the level of detection to 60 CFU/g. Formaldehyde had no effect on the level of detection with clam tissue. The level of detection of P shigelloides in oyster tissue without enrichment was 6 x 10(5) CFU/g. The addition of 4.0% formaldehyde to oyster tissue homogenates reduced the level of detection to 6 x 10(2) CFU/g in contrast to the addition of 0.1% BSA to PCR reactions or the DNA purification system which reduced the level of detection to only 2 x 10(5) CFU/g. The combination of formaldehyde plus BSA, formaldehyde plus DNA purification, or formaldehyde plus BSA plus DNA purification all gave a detection level of 2 x 10(2) CFU/g of oyster tissue. With clam tissue, the linear range for detection of P shigelloides was 60 to 2x 10(4) CFU/g. With oyster tissue, the linear range for detection of P shigelloides was 2 x 10(2) to 6x 10(4) CFU/g. (c) 2006 Elsevier B.V. All rights reserved.