Research - (2017) Volume 11, Issue 1
Biogenic amines are indispensable components of living cells; nevertheless these compounds could be toxic for human health in higher concentrations. Putrescine is supposed to be the major biogenic amine associated with microbial food spoilage. Development of reliable, fast and culture-independent molecular methods to detect bacteria producing biogenic amines deserves the attention, especially of the food industry in purpose to protect health.The objective of this study was to verify the newly designed primer sets for detection of two inducible genes adiAand speF together in Salmonella entericaand Escherichia coligenomeby Real-time PCR. These forenamedgenes encode enzymes inthe metabolic pathway which leads to production ofputrescine in Gram-negative bacteria. Moreover, relative expression of these genes was studied in E.coli CCM 3954strainusing Real-time PCR.In this study, sets of new primers for the detection two inducible genes (speFand adiA) in Salmonella entericaand E. coliby Real-time PCR were designed and tested. Amplification efficiency of a Real-time PCR was calculated from the slope ofthe standard curves (adiA, speF, gapA). An efficiency in a range from 95 to 105 % for all tested reactionswas achieved. Thegene expression (R) of adiAand speFgenes inE. coliwas varied depending on culture conditions. The highest gene expression of adiAand speFwas observed at 6, 24 and 36 h (RadiA ~ 3, 5, 9; RspeF~11, 10, 9; respectively) after initiation ofgrowth of this bacteria in nutrient brothmediumenchired with amino acids. The results show that these primers could be used for relative quantification analysis ofE. coli.