Rapid quantification of total bacteria on cod fillets by using real-time PCR

Abstract

Real-time PCR assays, based on universal primers and SYBR® Green technology, were applied tothe 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 derivedfrom 1 x 101 to 1 x 105 CFU/real-time PCR assay. Real-time-PCR allowed construction of astandard curve that facilitated quantification of total bacteria from fish fillets. Differential cen-trifugation and washing of tissue homogenate pellets was successful in eliminating PCR inhibi-tion by tissue components. There was no statistically significant difference (P >0.01) betweenplate counts from cod fillets and counts derived from real-time PCR. The minimum detectionlimit with cod tissue was target DNA derived from 10 CFU/real-time PCR equivalent to 1 x 105CFU/g. The real-time PCR assay proved to be a rapid and reliable assay for quantification of totalbacteria on fish fillets.