Rapid detection and quantification of tyrosine decarboxylase gene (tdc) and its expression in gram-positive bacteria associated with fermented foods using PCR-based methods.
In this study, PCR-based procedures were developed to detect the occurrence and quantify the expression of the tyrosine decarboxylase gene (tdc) in gram-positive bacteria associated with fermented foods. Consensus primers were used in conventional and reverse transcription PCR to analyze a collection of 87 pure cultures of lactic acid bacteria and staphylococci. All enterococci, Staphylococcus epidermidis, Lactobacillus brevis, Lactobacillus curvatus, and Lactobacillus fermentum strains and 1 of 10 Staphylococcus xylosus strains produced amplification products with the primers DEC5 and DEC3 in accordance with results of the screening plate method and with previously reported result obtained with high-performance liquid chromatography. No amplicons were obtained for tyramine-negative strains, confirming the high specificity of these new primers. A novel quantitative real-time PCR assay was successfully applied to quantify tdc and its transcript in pure cultures and in meat and meat products. This assay allowed estimation of the influence of different variables (pH, temperature, and NaCl concentration) on the tdc expression of the tyraminogenic strain Enterococcus faecalis EF37 after 72 h of growth in M17 medium. Data obtained suggest that stressful conditions could induce greater tyrosine decarboxylase activity. The culture-independent PCR procedures developed here may be used for reliable and fast detection and quantification of bacterial tyraminogenic activity without the limitations of conventional techniques.
Torriani
Sandra
S
Dipartimento Scientifico e Tecnologico, Università degli Studi di Verona, Strada Le Grazie 15, 37134 Verona, Italy. sandra.torriani@univr.it
Gatto
Veronica
V
Sembeni
Silvia
S
Tofalo
Rosanna
R
Suzzi
Giovanna
G
Belletti
Nicoletta
N
Gardini
Fausto
F
Bover-Cid
Sara
S
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D005285:Fermentation; D005506:Food Contamination / Q000032:analysis*; D005516:Food Microbiology; D005784:Gene Amplification; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D006094:Gram-Positive Bacteria / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D008461:Meat Products / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012965:Sodium Chloride / Q000494:pharmacology; D013696:Temperature; D014439:Tyramine / Q000096:biosynthesis; D014445:Tyrosine Decarboxylase / Q000235:genetics* / Q000378:metabolism*
D012965:Sodium Chloride; D014445:Tyrosine Decarboxylase; D014439:Tyramine