Detection of Viable Vibrio mimicus by Reverse Transcription-Polymerase Chain Reaction

Abstract

Differentiating viable cells from nonviable cells is of considerable importance in the monitoring of food-borne pathogens. A reverse transcription-polymerase chain reaction (RT-PCR) assay was developed to detect mRNA from the phospholipase gene (phl) of Vibrio mimicus. Viable V. mimicus cells were killed by heat or ethanol treatment and kept for various periods at room temperature. Total RNA from V. mimicus was extracted, treated with DNase and subjected to RT-PCR with primers for the phl gene. The phl mRNA was detected in the viable cells, but it gradually disappeared when the killed cells were left at room temperature and became undetectable after 8 h of storage. Furthermore, RT-PCR generated a 493 by fragment from the total RNA extracted from as few as about 10³ organisms, confirming the sensitivity of the assay. The amplification of the phl mRNA was specific for V. mimicus, as no amplification was found when fifteen other Vibrio species and seven related organisms were tested. The results indicated a good relationship between the detection of the phl mRNA and viability of V. mimicus cells because the phl transcript is rapidly degraded upon cell death. This work shows the usefulness of RT-PCR as a sensitive method for the specific detection of viable V. mimicus.