RESUMO
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.
Assuntos
Bactérias/isolamento & purificação , Contaminação de Alimentos/análise , Fator Tu de Elongação de Peptídeos/genética , Reação em Cadeia da Polimerase/métodos , Bactérias/genética , Contagem de Colônia Microbiana/métodos , Qualidade de Produtos para o Consumidor , DNA Bacteriano/análise , Microbiologia de Alimentos , Amplificação de Genes , Humanos , Modelos Lineares , Reação em Cadeia da Polimerase/normas , Sensibilidade e Especificidade , Especificidade da Espécie , Fatores de TempoRESUMO
The possession of a respiration-dependent primary sodium pump and the requirement of Na for growth were investigated in bacterial isolates from marine environments. The bacteria in which NADH oxidase specifically required Na for maximum activity were believed to possess a primary sodium pump. All bacteria that failed to grow without the addition of NaCl possessed a primary Na pump. All bacteria that had no primary Na pump grew without additional NaCl. The primary Na pump seems to be involved in the Na requirement of marine bacteria, and this can be regarded as a criterion for the definition of marine bacteria.
