Development of molecular methods for quantitation of Plesiomonas shigelloides in seafood

Plesiomonas shigelloides has been recognized as a potential human and animal pathogen in the past six decades. In this study, RAPD typing was performed to determine the genetic variability among the isolates of P. shigelloides from different sources. In addition, quantitative methods to detect P. shigelloides based on PCR were developed. Also, factors affecting PCR detection were examined, methods to reduce the inhibition of PCR by PCR inhibitors were tested, and a method for quantitative detection of viable P. shigelloides in a mixture of viable and dead cells was developed.; RAPD analysis showed that there was notable genetic variability among most of the strains of P. shigelloides isolates from fresh water, fish and human clinical sources. The isolates from fish had a closer linkage to the human clinical isolates than did the freshwater isolates, suggesting that fish may be the more serious source for potential risk of infection.; Using standard quantitative PCR, the lowest level of detection for P. shigelloides, without enrichment, was 60 CFU/g in clams with the treatment of BSA and 200 CFU/g in oysters with the treatment of formaldehyde and BSA respectively.; Using conventional quantitative PCR in combination with ethidium bromide monoazide (EMA) enabled quantitative detection of viable P. shigelloides cells in varying ratio's of viable to dead cells at a minimum of 24 CFU per PCR reaction.; Competitive PCR was capable of detecting DNA derived from 2.4 x 102 to 2.4 x 105 CFU/g of clam tissue without enrichment, and 4.0 x 101 to 1.2 x 104 CFU/g of clam tissue with 7 hrs. of nonselective enrichment at 37°C respectively.; Real-time PCR quantitatively detected 3 to 1.0 x 104 CFU per gram of oyster after 7 hrs. nonselective enrichment. Without enrichment, the minimum detection level was 1.0 x 103 CFU per gram of oyster tissue with the addition of formaldehyde following by the treatment with coated charcoal.