Development of a quantitative real-time PCR method to enumerate total bacterial counts in ready-to-eat fruits and...

A newly developed real-time PCR assay rapidly quantifies the total bacterial numbers in contaminated ready-to-eat vegetables and fruits compared with the standard plate count method. Primers targeting the rpoB gene, which encodes for the beta subunit of the bacterial RNA polymerase and which is common to most bacterial species, was used instead of the 16S rRNA gene, which has multiple copies and varies among bacterial species. A primer pair specific for rpoB was confirmed to amplify rpoB in a wide range of bacterial species after we assessed 49 strains isolated from five kinds of fruits and vegetables. We purchased fruits and vegetables from retail shops and enumerated the bacteria associated with them by use of real-time PCR and compared this to the number found by the culture method. We found a high correlation between the threshold PCR cycle number when compared with the plate count culture number. The real-time PCR assay developed in this study can enumerate the dominant bacterial species in ready-to-eat fruits and vegetables.