Levin E Robert
Real-time PCR assays, based on universal primers and SYBR® Green technology, were applied to the quantification of total bacteria derived from cod tissue. The detection range of the real time-PCR assay for 16S rDNA sequences from mixed bacterial cultures was linear for DNA derived from 1 x 101 to 1 x 105 CFU/real-time PCR assay. Real-time-PCR allowed construction of a standard curve that facilitated quantification of total bacteria from fish fillets. Differential centrifugation and washing of tissue homogenate pellets was successful in eliminating PCR inhibition by tissue components. There was no statistically significant difference (P >0.01) between plate counts from cod fillets and counts derived from real-time PCR. The minimum detection limit with cod tissue was target DNA derived from 10 CFU/real-time PCR equivalent to 1 x 105 CFU/g. The real-time PCR assay proved to be a rapid and reliable assay for quantification of total bacteria on fish fillets.
