Detection of Yersinia enterocolitica in pure culture by PCR using internal (IAC) and external (EAC) amplification controls.

MASTRODONATO ANNA CHIARA; LUCERO ESTRADA CECILIA; FAVIER GABRIELA; DI MARCO NATALIA; ESCUDERO MARIA ESTHER

San Miguel de Tucumán

Congreso; XII Congreso Argentino de Microbiología General (SAMIGE); 2017

Yersinia enterocolitica, an enterobacterium present in certain foods, can cause infections in humans. Its detection 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 false negative results, it is advisable to introduce a competitive internal amplification control (IAC) that is co-amplified with the target DNA sequence by the same primer pair in PCR. The IAC and target DNA products (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 primers with the target gene. In contrast with positive samples where two bands (16S rDNA and IAC) will be observed, negative samples will show only one band corresponding to IAC. In the present study, we included an IAC previously designed by this research team, in a well-known PCR protocol to amplify the 16S rDNA gene from Y. enterocolitica. The IAC was introduced in the PCR reaction and amplified when Y. enterocolitica cultures was assayed. When Y. enterocolitica concentration was 4.5 x 103 CFU ml-1 in pure culture, the IAC at concentration of 2.94 ag µl-1 in the PCR reaction mixture was co-amplified with the 16S rDNA sequence, producing bands of 710 and 300 bp, respectively. These Y. enterocolitica values were considered the detection limits of the duplex PCR. Furthermore, an external amplification control (EAC) which has the same size as 16S rDNA amplicon (300 bp) was amplified in parallel as a positive control regarding future studies where unknown samples will be analyzed. The specific detection of Y. enterocolitica by PCR including IAC and EAC might be achieved directly on food samples when the pathogen load reaches concentrations of at least 4.5 x 103 CFU ml-1.