Design of a competitive internal amplification control (CIAC) for PCR targeted to Yersinia enterocolitica-yst gene.

MASTRODONATO, ANNA CHIARA; LAPADULA, WALTER; MAXIMILIANO JURI AYUB; FAVIER GABRIELA ISABEL; LUCERO ESTRADA CECILIA; ESCUDERO MARÍA ESTHER

Salta

Congreso; LV Reunión anual de la Sociedad Argentina de Bioquímica y Biología Molecular, SAIB;XIV PABMB Congress (Panamerican Association of Biochemistry and Molecular Biology); 2019

Sociedad Argentina de Bioquímica y Biología Molecular

The isolation of Yersinia enterocolitica (Ye) from complex matrices such as foods presents difficulties due to the competition between this microorganism and the accompanying flora. To overcome the limitations related to culture techniques, molecular methods based on polymerase chain reaction (PCR) have been developed to detect virulence genes such as yst (Ye thermostable toxin). However, foods are complex matrices that may contain inhibitors of this reaction. To avoid false negative results, it is advisable to introduce a competitive internal amplification control (cIAC) into the PCR reaction mixture. This is a DNA molecule which is amplified by the same primers as the target sequence, but that is distinguished from it by its different molecular size. Thus, the cIAC is incorporated into the PCR reaction mixture where, together with the target DNA, will compete for the primers. In yst negative samples, one band corresponding to cIAC should be observed, unlike two bands (yst and cIAC) that will be observed in a positive sample. To 3 obtain the cIAC, a pair of oligonucleotides complementary to a DNA region belonging to the Culex molesus genome and flanked at their ends by the primer sequences targeted to Ye yst gene, were designed. This C. molesus sequence was analyzed by BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) to verify the absence of regions complementary to the yst gene. The cIAC was amplified by PCR using Taq polymerase and subsequently cloned into the pGEM-T Easy® vector (pG, Invitrogen), obtaining a product of 417 bp (greater molecular weight than the yst amplicon of 190 bp). The cIAC will allow check the efficiency of yst PCR in the detection of Ye in foods.