A Novel Method of Extraction and Purification of Bacterial DNA for PCR Detection of MecA, 16SrRNA, VanB

Raheem Hashim Abdullah; Adil Abaed Hassoni

doi:10.61796/jmgcb.v1i4.403

Authors

Raheem Hashim Abdullah
rheemalhy93@gmail.com
Department of Medical Laboratory Techniques, College of Health and Medical Techniques, Al-Furat Al-Awsat Technical University, Kufa, Iraq
Adil Abaed Hassoni ALmussiab Technical College, AL-Furat AlAwsat Technical University, Iraq

Vol. 1 No. 4 (2024): Journal of Medical Genetics and Clinical Biology

Articles

April 25, 2024

Downloads

View Article

Abstract
How to Cite
Metrics
References
License

Bacteria are the main pest of nosocomial infections in healthcare facilities around the world. The aim of this study is to modify and minimize the amount of time, while still having no influence on the extraction's purity of DNA the method of extracting the DNA molecule and compare it with other methods that take longer to extract and purify DNA. This study examined two processes for isolating DNA from Gram-positive bacterial species: a boiling method and an enzymatic method that took one hour. In addition, the collected DNA was subjected to spectrophotometry, agarose gel electrophoresis, and PCR for both quantitative and qualitative examination. The results presented in this work show that boiling method pretreatment facilitates efficient cell lysis and DNA extraction, leading to increased yields of isolated bacterial DNA. In addition, DNA suitability for PCR-based identification of mecA,16SrRNA, VanB to Gram positive S. aureus strains was analyzed. Our findings demonstrated that the DNA generated by this simple approach is low-cost, quick, and safe, and that the process may be utilized in PCR methods on a wide range of gram-positive and gram negative species, as well as in laboratories without the necessary materials, tools, or technology

Abdullah, R. H., & Hassoni, A. A. (2024). A Novel Method of Extraction and Purification of Bacterial DNA for PCR Detection of MecA, 16SrRNA, VanB . Journal of Medical Genetics and Clinical Biology, 1(4), 43–51. https://doi.org/10.61796/jmgcb.v1i4.403

Download Citation

M. F. Cotton, E. Wasserman, J. Smit, A. Whitelaw, and H. J. Zar, “High incidence of antimicrobial resistant organisms including extended spectrum beta-lactamase producing Enterobacteriaceae and methicillin-resistant Staphylococcus aureusin nasopharyngeal and blood isolates of HIV-infected children from Cape Town, South ,” BMC Infect. Dis., vol. 8, no. 1, pp. 1–10, 2008.

A. R. Najm, I. O. Pokudina, A. H. Abd Ali, D. Alset, and T. P. Shkurat, “Study genetic polymorphisms IL-1β, IL-28 and susceptibility to antibiotics of Staphylococcus aureus in children with recurrent respiratory infections,” Gene Reports, vol. 27, p. 101593, 2022.

M. A. Pesquero, L. C. Carneiro, and D. D. J. Pires, “Insect/Bacteria association and nosocomial infection,” Salmonella, A Divers. Superbug, pp. 449–468, 2012.

K. V Chaitanya and K. V Chaitanya, “Structure and organization of virus genomes,” Genome Genomics From Archaea to Eukaryotes, pp. 1–30, 2019.

A. N. H. Creager, “Recipes for recombining DNA: a history of molecular cloning: a laboratory manual,” BJHS Themes, vol. 5, pp. 225–243, 2020.

B. A. Omar, H. A. Atif, and M. E. Mogahid, “Comparison of three DNA extraction methods for polymerase chain reaction (PCR) analysis of bacterial genomic DNA,” African J. Microbiol. Res., vol. 8, no. 6, pp. 598–602, 2014.

C. Li, Y. Yang, and L. Ren, “Genetic evolution analysis of 2019 novel coronavirus and coronavirus from other species,” Infect. Genet. Evol., vol. 82, p. 104285, 2020.

I. Stojan, Ž. Trumbić, I. Lepen Pleić, and D. Šantić, “Evaluation of DNA extraction methods and direct PCR in metabarcoding of mock and marine bacterial communities,” Front. Microbiol., vol. 14, p. 1151907, 2023.

S. Fleige and M. W. Pfaffl, “RNA integrity and the effect on the real-time qRT-PCR performance,” Mol. Aspects Med., vol. 27, no. 2–3, pp. 126–139, 2006.

K. H. Wilson, R. B. Blitchington, and R. C. Greene, “Amplification of bacterial 16S ribosomal DNA with polymerase chain reaction,” J. Clin. Microbiol., vol. 28, no. 9, pp. 1942–1946, 1990.

T. J. Boltje, T. Buskas, and G.-J. Boons, “Opportunities and challenges in synthetic oligosaccharide and glycoconjugate research,” Nat. Chem., vol. 1, no. 8, pp. 611–622, 2009.

B. Aaliya et al., “Recent trends in bacterial decontamination of food products by hurdle technology: A synergistic approach using thermal and non-thermal processing techniques,” Food Res. Int., vol. 147, p. 110514, 2021.

N. Gupta, “DNA extraction and polymerase chain reaction,” J. Cytol., vol. 36, no. 2, pp. 116–117, 2019.

M. Samuel, M. Lu, C. J. Pachuk, and C. Satishchandran, “A spectrophotometric method to quantify linear DNA,” Anal. Biochem., vol. 313, no. 2, pp. 301–306, 2003.

M. N. Hoque et al., “Antibiogram and virulence profiling reveals multidrug resistant Staphylococcus aureus as the predominant aetiology of subclinical mastitis in riverine buffaloes,” Vet. Med. Sci., vol. 8, no. 6, pp. 2631–2645, 2022.

“Warm ocean anomaly, air sea fluxes, and the rapid intensification of tropical cyclone Nargis (2008),” Geophys. Res. Lett., vol. 36, no. 3, 2009.

J. R. Johnson and A. L. Stell, “Extended virulence genotypes of Escherichia coli strains from patients with urosepsis in relation to phylogeny and host compromise,” J. Infect. Dis., vol. 181, no. 1, pp. 261–272, 2000.

S. Yamamoto, A. Terai, K. Yuri, H. Kurazono, Y. Takeda, and O. Yoshida, “Detection of urovirulence factors in Escherichia coli by multiplex polymerase chain reaction,” FEMS Immunol. Med. Microbiol., vol. 12, no. 2, pp. 85–90, 1995.

M. Dairawan and P. J. Shetty, “The evolution of DNA extraction methods,” Am. J. Biomed. Sci. Res, vol. 8, no. 1, pp. 39–45, 2020.

C. A. Lewis, “Organic Extraction of Nucleic Acids Using Ethanol Precipitation or Microcon® Centrifugal Filter Purification Methods,” in Forensic DNA Analysis: Methods and Protocols, Springer, 2023, pp. 23–33.

T. C. Glenn, “Field guide to next‐generation DNA sequencers,” Mol. Ecol. Resour., vol. 11, no. 5, pp. 759–769, 2011.

D. Aphale and A. Kulkarni, “Modifications and optimization of manual methods for polymerase chain reaction and 16S rRNA gene sequencing quality community DNA extraction from goat rumen digesta,” Vet. World, vol. 11, no. 7, p. 990, 2018.

C. James, R. Dixon, L. Talbot, S. J. James, N. Williams, and B. A. Onarinde, “Assessing the Impact of Heat Treatment of Food on Antimicrobial Resistance Genes and Their Potential Uptake by Other Bacteria—A Critical Review,” Antibiotics, vol. 10, no. 12, p. 1440, 2021.

G. H. Ali and N. L. Seiffein, “Association of some virulence genes in Methicillin resistant and Methicillin sensitive Staphylococcus aureus infections isolated in community with special emphasis on pvl/mecA genes profiles in Alexandria, Egypt,” Gene Reports, vol. 25, p. 101334, 2021.

O. B. Ahmed and A. S. Dablool, “Quality improvement of the DNA extracted by boiling method in gram negative bacteria,” Int. J. Bioassays, vol. 6, no. 4, pp. 5347–5349, 2017.

S. Merk, H. Meyer, I. Greiser‐Wilke, L. D. Sprague, and H. Neubauer, “Detection of Burkholderia cepacia DNA from Artificially Infected EDTA‐blood and Lung Tissue Comparing Different DNA Isolation Methods,” J. Vet. Med. Ser. B, vol. 53, no. 6, pp. 281–285, 2006.