Development of a Quantitative Real-time PCR Method to Determine the Total Bacterial Count in Fresh Poultry Meat

A real-time PCR approach was developed to determine the total bacterial count in fresh poultry meat comparing the results with standard plate count technique. An initial nucleotide sequence analysis based on 16S rRNA PCR fragment was performed to estimate the bacterial diversity in poultry meat. 71 bacterial strains were identified among which the bacterial family Enterobacteriaceae and the genus Pseudomonas, and Lactobacillus were found to be predominant. The rpoB gene, encoding the 8,subunit of RNA polymerase, which has only one copy in the bacterial genome, was selected as an amplification target. The "total viable count (TVC)" of commercially obtained poultry meat samples was determined using standard plate count method. A DNA isolation process was performed subsequently. The primer set designed to target the highly conserved regions on the rpoB gene was applied to detect a wide range of bacteria on poultry meat samples. Using the "SYBR (R) Green l" system, a melting temperature analysis was performed to confirm the amplification specificity of the primer. The comparison of C-t-values obtained by real-time PCR analysis with the bacterial count resulting from the standard plate count method (Log(10), CFU/g) showed a good correlation (R-2 = 0.89, y = -2.73x + 47.52) over the range of 10(3) to 10(9) CFU/g of sample tissue. The student's t-test has shown no significant difference between the results of the two methods (p<0.001). The real-time FOR assay developed in this study has shown a robust potential to determine the total bacterial count on poultry meat.