BIOLOGICAL SAMPLES CONTAMINATION CONTROL OF THE SUS SCROFA USING HAPLOID DNA MARKERS

Abstract

This paper proposes an effective method for controlling the contamination of biological samples of Sus scrofa with alien material in the preanalytical phase of a PCR study. Because PCR is highly sensitive, even a small amount of DNA containing alien biological substances can lead to false results. In the case of analysis of contaminated biological samples using diploid DNA markers, a mixture of two different homozygotes will be defined as a heterozygote. Unlike diploid DNA markers, a mixture of two different haplotypes is uniquely determined. To perform the study in the slaughter shop of the Globinsky Meat Processing Plant, after slaughter, one ear was cut off from the carcasses of pigs with an animal identification number tag. DNA was isolated from the epithelial tissue of the auricle. Five SNPs of the mitochondrial genome were used as a haploid marker. The study was carried out using a multisite PCR-RFLP method, the peculiarity of which was the analysis of the D-loop fragment between positions 15531 and 15959 of the porcine mitochondrial genome (GenBank: AJ002189.1). This sequence contains one monomorphic site (15558W) and five polymorphic Tas I restriction enzyme sites (15616T > C, 15714T > C, 15758T > C, and 15916A > T). The presence or absence of the Tas I site in the above positions determines mitochondrial haplotypes, denoted by Latin letters from A to P. PCR-RFLP analysis of DNA samples revealed DNA fragments on the electrophoregram, indicating a mixture of two or more haplotypes. It was possible to establish the presence of contamination due to the use of a multisite PCR-RFLP method, which implies a strictly discrete set of restrictive fragments for the mitochondrial DNA of an individual animal. The total size of the restriction fragments should be 428 bp and the presence of additional DNA bands indicate the association of two or more haplotypes. Thus, it was demonstrated that the use of haploid DNA markers makes it possible to determine the contamination of samples with alien material. This method can be used in the study of porcine nuclear DNA as a laboratory quality assurance of the preanalytical phase, which will reduce laboratory costs, improve the organization of work and avoid dramatic errors when performing genetic examinations. Keywords: pigs, epithelium, mitochondrial genome, haploid DNA markers, PCR, contamination.