Design of a new universal real-time PCR system targeting the tuf gene for the enumeration of bacterial counts in food.
A novel universal real-time PCR, consisting of newly designed oligonucleotide subsets, was designed for a bacterial housekeeping gene encoding the peptide elongation factor Tu. Specificity and universality were confirmed in 66 bacterial strains, including 51 genera and 63 species. The amplification kinetics of tuf gene-targeted real-time quantitative PCR were consistent in a wide range of bacterial species tested. A calibration curve (r(2) = 0.97) was produced for the estimation of bacterial counts, based on measurements of representative inoculations with 10-fold serial dilutions of the cells of representative bacterial species. Linear regression analysis of the real-time PCR-derived bacterial counts and aerobic plate counts, in a total 149 samples consisting of 25 minced meat, 34 fresh-cut vegetables, and 90 fish, exhibited a high correlation (r(2) = 0.84, 0.87, and 0.95, respectively) over the range of 3.0 to 9.0 log CFU/g. In total, the difference between the two methods was less than 0.5 log in 75 of these samples, and in the remaining 74 samples, the difference was 0.5 to 1.0 log. Presently, our tuf gene-targeted real-time quantitative PCR assay achieves a rapid (within 2 h) estimation of bacterial counts of 3.0 to 9.0 log CFU/g, in a practical manner.
Tanaka
Yuichiro
Y
Department of Food Science and Technology, Faculty of Marine Science, Tokyo University of Marine Science and Technology, Minato, Tokyo, Japan.
Takahashi
Hajime
H
Simidu
Usio
U
Kimura
Bon
B
D001419:Bacteria / Q000235:genetics / Q000302:isolation & purification*; D015169:Colony Count, Microbial / Q000379:methods; D003257:Consumer Product Safety; D004269:DNA, Bacterial / Q000032:analysis; D005506:Food Contamination / Q000032:analysis*; D005516:Food Microbiology; D005784:Gene Amplification; D006801:Humans; D016014:Linear Models; D010444:Peptide Elongation Factor Tu / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012680:Sensitivity and Specificity; D013045:Species Specificity; D013997:Time Factors
D004269:DNA, Bacterial; D010444:Peptide Elongation Factor Tu