Selective Detection of Mixed Bacterial Survivors from Fish Fillets after Freezing and Thawing by Ethidium Bromide Monoazide Real-Time PCR

Abstract

The effect of freezing and thawing on the survival of mixed bacteria from cod fillets was studied using ethidium bromide monoazide (EMA) real-time PCR (EMA Rti-PCR). The permeability of cell membranes, media selection, EMA treatment, addition of glycerol, and the number of freeze-thaw cycles were investigated to discriminate and enumerate viable from dead bacteria following freezing and thawing. Universal primers were used to amplify the sequence of a conserved region of all eubacterial 16S rDNA for EMA Rti‐PCR. Amplification of DNA from frozen-thawed bacterial cells was significantly inhibited by EMA, whereas the DNA without EMA from frozen-thawed cells was readily amplified. Freezing and thawing resulted in damage to the cell membrane resulting in increased EMA uptake as revealed by confocal microscopy. The effect of glycerol on survival of the frozen-thawed cells was determined by plate counts and EMA Rti-PCR. Mixed bacteria from fish fillets suspended in saline containing glycerol (5–20%) resulted in greater survival following freezing and thawing than when bacteria were suspended in saline alone during freezing and thawing. Increasing the number of freeze-thaw cycles resulted in a decrease in the log CFU and an increase in Ct value.

Keywords:

• bacterial survivors
• CFU
• cod fillets
• EMA
• EMA-Rti-PCR
• Ethidium bromide monoazide
• freezing and thawing
• mixed bacteria
• plate counts
• real-time PCR
• Rti-PCR

ACKNOWLEDGMENTS

This study was supported by a special research grant from the USDA #2006–34423–17164.