Quantitative Real-time PCR detection of putrescine-producing Gram-negative bacteria

• Main Authors: Kristýna Maršálková, Khatantuul Purevdorj, Petra Jančová, Hana Pištěková, Leona Buňková
• Format: Article
• Language: English
• Published: HACCP Consulting 2017-01-01
• Series: Potravinarstvo
• Subjects: putrescine; Gram-negative bacteria; speF; adiA; Real-time PCR
• Online Access: http://www.potravinarstvo.com/journal1/index.php/potravinarstvo/article/view/739

<p>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 <em>adiA</em> and <em>speF </em>together in <em>Salmonella enterica</em> and <em>Escherichia coli </em>genome<em> </em>by Real-time PCR. These forenamed genes encode enzymes in&nbsp;the metabolic pathway which leads to production of putrescine in Gram-negative bacteria. Moreover, relative expression of these genes was studied in <em>E.&nbsp;coli </em>CCM 3954 strain using Real-time PCR. In this study, sets of new primers for the detection two inducible genes (<em>speF</em> and <em>adiA</em>) in <em>Salmonella enterica</em> and <em>E. coli</em> by Real-time PCR were designed and tested. Amplification efficiency of a Real-time PCR was calculated from the slope of&nbsp;the standard curves (<em>adiA</em>, <em>speF</em>, <em>gapA</em>). An efficiency in a range from 95 to 105 % for all tested reactions was achieved. The gene expression (R) of <em>adiA</em> and <em>speF</em> genes in<em> E. coli</em> was varied depending on culture conditions. The highest gene expression of <em>adiA</em> and <em>speF</em> was observed at 6, 24 and 36 h (R<em>_adiA</em>~ 3, 5, 9; R<em>_speF</em> ~11, 10, 9; respectively) after initiation of&nbsp;growth of this bacteria in nutrient broth medium enchired with amino acids. The results show that these primers could be used for relative quantification analysis of<em> E</em>. <em>coli</em>.</p>