Detection of Yersinia enterocolitica (ye) by 16s rDNA PCR in artificially contaminated meat, including amplification controls

ESCUDERO MARÍA ESTHER; FAVIER GABRIELA ISABEL; MASTRODONATO, ANNA CHIARA; LUCERO ESTRADA CECILIA STELLA MARYS

Merlo

Jornada; XXXV Reunión Anual de la Sociedad de Biología de Cuyo; 2017

Sociedad de Biología de Cuyo

YE is an enteropathogen that causes intestinal and immunological complications. It is transmitted by oral route, commonly by contaminated foods, with pigs as the main reservoirs. YE isolation from complex matrices such as food is complicated because of the competition between this microorganism and the accompanying microbiota. To overcome the limitations related to culture techniques, molecular methods based on polymerase chain reaction (PCR) have been developed, which are characterized by their rapidity, high specificity and sensitivity. The objective of this study was to detect YE at different dilutions in a ground beef sample by PCR targeted to the species-specific 16S rDNA gene including an internal amplification control (IAC) and an external amplification control (EAC). YE was cultured 24 h in trypticase soy broth (OD600 0.2) from which serial decimal dilutions corresponding to 108 to 100 CFU/ml were performed. Then, nine 10-g portions of one ground beef sample were contaminated with 0.4 ml of each inoculum dilution, submerged in 90 ml sorbitol bile peptone broth and incubated 24 h at 25°C to stimulate the YE repair in this complex matrix. DNA extraction was performed by boiling, and then, 16S rDNA PCR including IAC and EAC was performed. Amplification of the 16S rDNA gene was observed in all dilutions tested, while IAC was observed from YE concentration of 104 CFU/ml. EAC worked perfectly. The present study highlights the excellent performance of the amplification controls designed in our laboratory for the detection of YE when this bacterium is present in complex matrices.