pcr Standardization to Identify Lawsonia intracellularis in Pigs
Universidad Cooperativa de Colombia. Sede Bucaramanga.
Universidad Cooperativa de Colombia. Sede Bucaramanga.
Universidad Cooperativa de Colombia. Sede Bucaramanga.
Universidad Cooperativa de Colombia. Sede Bucaramanga.
Universidad Cooperativa de Colombia. Sede Bucaramanga.
Universidad Cooperativa de Colombia. Sede Bucaramanga.
Universidad Cooperativa de Colombia. Sede Bucaramanga.
Introduction: the nested Polymerase Chain Reaction (pcr)technique has been standardized for the diagnosis of porcine proliferative enteropathy caused by Lawsonia intracellularis. Methods: such technique was based upon amplifying a region of 270 pb of the 16s arnr gene of the Lawsonia intracellularis. For such purposes, adna sample was taken from the bacteria with a Purelink Genomic kit, Invitrogen ®, from 100 samples of porcine ileum of the metropolitan area of Bucaramanga (Santander). The dna samples were quantified with fluorometry and adjusted to 20 ng/μL concentration. The first amplification of the 319 pb part of the 16s gene was completed with the external initiators: 5’-tat ggc tgt caa aca ctc cg-3’ and 5’-tga agg tat tgg tat tct cc-3’. The result of this first amplification was used as a pattern for the second reaction, where the internal part of 270 pb of the 16s arn gene of L. intracellularis was amplified using the specific internal initiators: 5’- tta cag gtg aag tta ttg gg-3’ and 5’-ctt tct cat gtc cca taa gc-3’. The optimal conditions for the pcr for both internal and external initiators included 2,5 μm de mgcl2; 0,15 μm of each dntp; 1x of reaction buffer; 0,8 μm of each initiator; and 2,5u of Taq polymerase. As a positive control for the taken dna samples and the pcr, the bacterial dna taken from Enterisol ®(Boheringer Ingelheim®) was used. Results: optimal temperatures of annealing for the simple and nested pcr were 50 and 55°C, respectively. The detection limit of the technique was 5 fg. Conclusions: no cross reactions were found with Esquerichia coli, or with Salmonella typhimurium, which are bacteria with similar clinical conditions.
