Direct Application of the Polymerase Chain Reaction for Quantification of Total Bacteria on Fish Fillets

Abstract

A rapid, conventional PCR assay that quantifies total bacteria from fish fillets is described. The methodology is based on the use of the universal primers DG74 and RW01 to amplify a 370-bp conserved sequence of the 16S rRNA gene of Gram-positive and Gram-negative bacteria. The intensity of amplified DNA bands (from mixed bacterial flora of fish fillets) in agarose gels was found to be linear from 5 × 10² to 1 × 10⁵ CFU/PCR. Differential centrifugation of tissue homogenates followed by sample dilution was successful in eliminating PCR inhibition by tissue components. A standard curve was generated relating the intensity of fluorescence of DNA bands to the log of bacterial numbers for rapidly assessing the total number of bacteria per gram of fish tissue by PCR.

Key Words:

• PCR
• quantification
• total bacteria
• fish fillets

ACKNOWLEDGEMENTS

Jung-Lim Lee was supported in part by a postdoctoral fellowship grant from the Korea Research Foundation funded by the government of Korea (MOEHRD, Basic Research Promotion Fund, M01–2004–000–20192-0) and by a U.S.D.A Special Seafood Safety grant. This is paper no. 3376 from the Massachusetts Agricultural Experiment Station, University of Massachusetts, Amherst, Mass., USA.

Notes

^a+: strong signal detected, WS: weak signal, –: no signal detected, NT: not tested.

^bbold + used for quantification of total bacteria per gram of tissue.