Novel touchdown-PCR method for the detection of putrescine producing gram-negative bacteria in food products

Formation of biogenic amines may occur in food due to metabolic activities of contaminating Gram-negative bacteria. Putrescine is assumed to be the major biogenic amine associated with microbial food spoilage. Gram-negative bacteria can form putrescine by three metabolic pathways that can include eight different enzymes. The objective of this study was to design new sets of primers able to detect all important enzymes involved in the production of putrescine by Gram-negative bacteria. Seven new sets of consensual primers based on gene sequences of different bacteria were designed and used for detection of the speA, adiA, adi, speB, aguA, speC, and speF genes. A newly developed touchdown polymerase chain reaction (PCR) method using these primers was successfully applied on several putrescine-producers. Selected PCR products were sequenced and high similarity of their sequences (99-91%) with known sequences of the corresponding genes confirmed high specificity of the developed sets of primers. Furthermore, all the investigated bacteria produced both putrescine and agmatine, an intermediate of putrescine production, which was confirmed by chemical analysis. The developed new touchdown PCR method could easily be used to detect potential foodborne Gram-negative producers of putrescine. The newly developed sets of primers could also be useful in further research on putrescine metabolism in contaminating microbiota.