CONICET | Buscador de Institutos y Recursos Humanos

Yersinia enterocolitica, an enterobacterium present in certain foods, can cause infections in humans. Itsdetection in foods is possible by PCR targeted to the 16S rDNA gene using the primer pair 16SYerF-16SYerR.However, food samples are complex matrices and they might contain PCR inhibitors. To avoid falsenegative results, it is advisable to introduce a competitive internal amplification control (IAC) that isco-amplified with the target DNA sequence by the same primer pair in PCR. The IAC and target DNAproducts (16S rDNA amplicon) can be identified by their different molecular (710 and 300 bp, respectively).Thus, the IAC is incorporated into the PCR reaction mixture where will compete for primerswith the target gene. In contrast with positive samples where two bands (16S rDNA and IAC) will beobserved, negative samples will show only one band corresponding to IAC. In the present study, weincluded an IAC previously designed by this research team, in a well-known PCR protocol to amplify the16S rDNA gene from Y. enterocolitica. The IAC was introduced in the PCR reaction and amplified whenY. enterocolitica cultures was assayed. When Y. enterocolitica concentration was 4.5 x 103 CFU ml-1in pure culture, the IAC at concentration of 2.94 ag μl-1 in the PCR reaction mixture was co-amplifiedwith the 16S rDNA sequence, producing bands of 710 and 300 bp, respectively. These Y. enterocoliticavalues were considered the detection limits of the duplex PCR. Furthermore, an external amplificationcontrol (EAC) which has the same size as 16S rDNA amplicon (300 bp) was amplified in parallel as a positivecontrol regarding future studies where unknown samples will be analyzed. The specific detection ofY. enterocolitica by PCR including IAC and EAC might be achieved directly on food samples when thepathogen load reaches concentrations of at least 4.5 x 103 CFU ml-1.