DOI: 10.32900/2312-8402-2023-129-70-78
Keywords: pigs, epithelium, mitochondrial genome, haploid DNA markers, PCR, contamination
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.
References
- Pocherniaiev, K., F. (2017). Novi mozhlyvosti bahatosaitovoho sposobu vyznachennia mitokhondrialnykh haplotypiv svynei [New possibilities of the multi-site method of determination of mitochondrial haplotypes of pigs] Svynarstvo. Mizhvidomchy itematychnyinaukovy izbirnyk Instytutu svynarstva i APV NAAN. 69, 100-108 https://svinarstvo.com/zbirnyk/archive/69/69-100-108.pdf [inUkranian].
- Furutani, S., Nagai, H., Takamura, Y., Aoyamaa, Y., & Kubo, I. (2012). Detection of expressed gene in isolated single cells in microchambers by a novel hot cell-direct RT-PCR method. Analyst. Vol. 137. P. 2951-2957. https://doi.org/10.1039/C2AN15866C.
- Shunsuke, F., Naoki, S., Hidenori, N., Yuri, A., & Izumi,K. (2014). Development of a Detection System for Expressed Genes in Isolated Single Jurkat Cells. Sensors and Materials, 26(8), 623–635. URL: https://sensors.myu-group.co.jp/sm_pdf/SM1029.pdf
- Luyao, L., Xiaobin, D., Yunping, T., Guijun, M., Zhongping, Z., Lulu, Z., Zewen, W., Duli, Y., & Xianbo, Q. (2021). Methods and platforms for analysis of nucleic acids from single-cell based on microfluidics. Microfluidics and Nanofluidics, 25(87). https://doi.org/10.1007/s10404-021-02485-0.
- Ma, J., Tran, G., Wan, A., M., D., Young, E., W., K., Kumacheva, E., Iscove, N., N., Zandstra, & P., W. (2021). Microdroplet-based one-step RT-PCR for ultrahigh throughput single-cell multiplex gene expression analysis and rare cell detection. Scientific Reports, 11(1), 6777. https://doi.org/10.1038/s41598-021-86087-45.
- Povkh, A. S., & Romanchuk, S., M. (2018). Kontaminatsiia pid chas molekuliarno- henetychnoho doslidzhennia. Prychyny yii vynyknennia ta naslidky. [Contamination during molecular-genetic research. Its causes and consequences]. Forensis Herald, 30(2), 106–115. https://doi.org/10.37025/1992-4437/2018-30-2-106 [in Ukranian].
- Stepaniuk, R. K, & Ionova, V. V. (2020). Pryznachennia sudovoi molekuliarno-henetychnoi ekspertyzy na stadii dosudovoho rozsliduvannia: problemy ta shliakhy yikh vyrishennia. [The assignment of forensic molecular-genetic examination during pre-trial investigation: problems and ways to solve them]. Bulletin of Luhansk State University of Internal Affairs Named After E. Didorenko, 3(91), 307-319. https://doi.org/10.33766/2524-0323.91.307-319 [in Ukranian].
- Balk, C. (2015). Reducing Contamination in Forensic Science. Themis, 3, 222-239. https://doi.org/10.31979/THEMIS.2015.0312
- Angela, G., Scot, E., D., Susan, G., Brian, D., & Rodrick, J., C. (2016). Inherent bacterial DNA contamination of extraction and sequencing reagents may affect interpretation of microbiota in low bacterial biomass samples. Gut Pathogens, 8(24); https://doi.org/10.1186/s13099-016-0103-7
- Van Oorschot, R. A., H., Szkuta, B., Meakin, G. E., Kokshoorn, B., & Goray, M. (2019). DNA transfer in forensic science: A review. Forensic Science International: Genetics, 38; 140-166. https://doi.org/10.1016/j.fsigen.2018.10.014
- Georgina, E. M., Bas, K., Roland, A. H. O., & Bianca, S. (2021). DNA transfer in forensic science. WIREs Forensic Sci, 3(1404); 1-19. https://doi.org/10.1002/wfs2.1404
- Giovambattista, G., Ripoli, M. V., Lirón, J. P., Villegas Castagnasso, E. E., Peral-García P., & Lojo, M. M. (2001). DNA typing in cattle stealing case. J Forensic Sci. 46(6);1484-6.PMID:11714164
- Budowle, B., Garofano, P., Hellman, A., Ketchum, M., Kanthaswamy, S., Parson, W., et al. (2005). Recommendations for animal DNA forensic and identity testing. Int J. Legal Med. 119(5); 295-302.https://doi.org/10.1007/s00414-005-0545-9
- Vashchenko, P. A,, Balatsky, V. M., Pocherniaev, K. F., Voloshchuk, V. M., Tsybenko, V. H., Saenko, A. M., Oliynychenko,Ye. K., Buslyk, T. V., & Rudoman, H. S. (2019). Genetic characteristic of the Mirhorod pig breed by analysing single nucleotide polymorphisms. Agricultural Scienceand Practice. 6(2); 47-57. https://doi.org/10.15407/agrisp6.02.047.