Quantification of total viable bacteria on fish fillets by using ethidium bromide monoazide real-time polymerase...

Real-time PCR based on universal primers for amplification of a highly conserved bacterial 16S rDNA sequence was utilized in conjunction with the treatment of extracted bacterial cells with ethidium bromide monoazide (EMA) for the differential enumeration of viable and dead cells on cod fillets. Amplification of DNA from dead bacterial cells was successfully inhibited by EMA, whereas the DNA from viable cells was readily amplified. The detection range of the EMA real-time PCR assay was linear from 1 x 10(1) to 1 x 10(5) mixed bacterial genomic targets per PCR derived from broth cultures of fish tissue. The minimum detection limit of bacteria was found to be 1 x 10(1) genomic units/real-time PCR, equivalent to 1 x 10(5) CFU per gram of tissue. The EMA real-time PCR allowed construction of a standard curve obtained by plotting the log of genomic targets from strictly viable cells against resulting PCR cycles (Ct values) that facilitated quantification of total viable bacteria from fish fillets. The log of the total number of genomic DNA targets from EMA treated cells and plate counts from six randomly procured cod fillets were found not to be statistically different with the exception of one fillet. The process of freezing and thawing fillet tissue resulted in a drop in mean colony forming units (CFU) detected by plate counts from log 8.5+/-0.2 to log 8.1+/-0.1. A similar reduction in genomic targets from 8.5+/-0.1 to 8.0+/-0.16 was detected by EMA real-time PCR.