Development of a rapid PCR protocol to detect in clams.

is part of the natural microflora of estuarine and coastal marine waters and can be also present in seafood, especially shellfish and bivalve molluscs. In this study we compared the reference cultural method ISO 6887-3 with two molecular methods, multiplex PCR and real-time PCR, for the detection of two distinct genetic markers ( species-specific gene and virulence gene) of in bivalve mollusc. The analyses were performed on clams inoculated with ATCC 43996 at T0 and after a 3 and 6 h of pre-enrichment in alkaline saline peptone water. Counts on agar plates were largely inaccurate, probably due to other species grown on the TCBS selective agar. Multiplex PCR assays, performed using primers pairs for and genes, showed a detection limit of 10 CFU/g of shell stock within 6 h of pre-enrichment, respecting however the action level indicated by the National Seafood Sanitation Program guideline. Detection by gene in real-time PCR reached the definitely highest sensitivity in shorter times, 10 CFU/g after 3 h of pre-enrichment, while the sensitivity for the gene was not promising, detecting between 10 and 10 CFU/g after 6 h of pre-enrichment. Our findings provide a rapid routine method of detection of based on gene by real-time PCR for commercial seafood analysis to identify the risk of gastrointestinal diseases.