Fiziksel ve Kimyasal Koşulların Çeşitli Yöntemlerle Elde Edilmiş Viral DNA’ya Etkileri

DNA virusları tamir mekanizmaları ve RNA viruslarına göre daha stabil olmaları sayesinde çevre şartlarına dirençli bir yapı gösterirler. Sahip oldukları bu direnç mutasyonel değişiklikleri elimine ederek, virusun korunmuş bölgelerinin kolaylıkla amplifikasyonu sağlamasına rağmen, uygun olmayan laboratuvar koşulları ve inhibe edici ajanlar, viral DNA tespitine engel olabilmektedir. Viral DNA eldesinde çeşitli yöntemlerin karşılaştırılmasının yapıldığı bu çalışmada aynı zamanda farklı fiziksel ve kimyasal koşulların viral nükleik asite olan etkileri de sorgulandı. Bu amaçla biri konvansiyonel metot olmak üzere toplam dört farklı ekstraksiyon yöntemi bir parapoxvirus olan ecythma contagiosumun DNA’sını tespit amacıyla kullanıldı. Elde edilen DNA’lar verimlilikleri yönünden kıyaslandıktan sonra çeşitli fiziksel (basınç, UV) ve kimyasal (deterjan) koşullara maruz bırakıldı. Basınçlı buhar otoklavına maruziyetten sonra viral DNA amplifikasyonu gerçekleşirken; tween 20 ve uv radyasyon viral DNA amplifikasyonu inhibe ettiler. Ticari kitlerin çoğu DNA ekstraksiyonunun süresini kısaltmaya yardımcı olurken konvansiyonel yöntem viral DNA tespiti açısından en yüksek verimliliği sağladı.

Anahtar Kelimeler:

Ekstraksiyon, PCR, Viral DNA

The Effects of Physical and Chemical Conditions on Viral DNA Obtained by Various Methods

DNA viruses are more resistant to environmental conditions than RNA viruses due to their repair mechanisms and stablity of their genetic material. Despite these advantages, inadequate laboratory conditions and inhibitory agents can interfere with viral DNA detection. In this study, which compares various methods of viral DNA obtainment, the effects of different physical and chemical conditions on viral nucleic acid were also questioned. For this purpose, a total of four different extraction methods, one of which was the conventional method, have been used to detect the DNA of the parapox virus causing ecythma contagiosum. The isolated DNAs were exposed to various physical (pressure, uv) and chemical treatment (detergent) conditions before PCR amplification. While viral DNA amplification occured after exposure to autoclave pressure steam sterilizer; Tween 20 and UV radiation inhibited viral DNA amplification. While most commercial kits helped to shorten the duration of DNA extraction, the conventional method provided the highest efficiency for viral DNA detection.

Keywords:

Extraction, PCR, viral DNA,

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Akkutay-Yoldar Z, Oguzoglu TC, Akça Y, 2016: Diagnosis and phylogenetic analysis of orf virus in Aleppo and Saanen goats from an outbreak in Turkey. Virol Sin, 31(3), 270-273.
Arnal C, Ferre-Aubineau V, Besse B, Mignotte B, Schwartzbrod L, Billaudel S, 1999: Comparison of seven RNA extraction methods on stool and shellfish samples prior to hepatitis A virus amplification. J Virol Methods, 77(1), 17-26.
Besaratinia A, Pfeifer GP, 2012: Measuring the formation and repair of UV damage at the DNA sequence level by ligation-mediated PCR. In “DNA Repair Protocols”, Ed; Bjergbaek, L, Humana Press, Totowa, NJ, pp. 189-202.
Bickley JA, Hopkins DA, 1999: Inhibitors and enhancers of PCR. In “Analytical Molecular Biology: quality and validation”, Ed; Saunders GC and Parkers HC., pp.81-102.
Chacon-Cortes D, Griffiths LR, 2014: Methods for extracting genomic DNA from whole blood samples: current perspectives. J Biorep Sci App Med, 2, 1-9.
Choi WS, Rodríguez RA, Sobsey MD, 2014: Persistence of viral genomes after autoclaving. J Virol Methods, 198, 37-40.
Deng MY, Wang H, Ward GB, Beckham TR, McKenna TS, 2005: Comparison of six RNA extraction methods for the detection of classical swine fever virus by real-time and conventional reverse transcription–PCR. J Vet Diagn Invest, 17(6), 574-578.
Eischeid AC, Meyer JN, Linden KG, 2009: UV disinfection of adenoviruses: molecular indications of DNA damage efficiency. Appl Environ Microbiol, 75(1), 23-28.
Eskandani M, Hamishehkar H, Ezzati Nazhad Dolatabadi J, 2013: Cyto/Genotoxicity study of polyoxyethylene (20) sorbitan monolaurate (tween 20). DNA Cell Biol, 32(9), 498-503.
Inoshima Y, Morooka A, Sentsui H, 2000: Detection and diagnosis of parapoxvirus by the polymerase chain reaction. J Virol Methods, 84, 201–208.
Knowles DP, 2011: Poxviridae. In “Fenner's Veterinary Virology”, 4th Eds., Academic Press, Elsevier, Ed; N. Maclachlan N, Dubovi EJ, pp. 151-65.
Murphy FA, Gibbs EPJ, Horzinek MC, Studdert MJ, 1999: Veterinary Virology. Third ed. San Diego: Academic Press, pp. 335-342.
Reha-Krantz LJ, 2010: DNA polymerase proofreading: Multiple roles maintain genome stability. Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics, 1804(5), 1049-1063.
Sambrook J, Russell DW, 2001: Molecular cloning: a laboratory manual. Cold Spring Harbor: Cold Spring Harbor Laboratory Press.
Scherer K, Johne R, Schrader C, Ellerbroek L, Schulenburg J, Klein G, 2010: Comparison of two extraction methods for viruses in food and application in a norovirus gastroenteritis outbreak. J Virol Methods, 169(1), 22-27.
Skinner MA, Buller RM, Damon IK, Lefkowitz EJ, McFadden G, Mc Innes CJ, Mercer AA, Moyer RW, Upton C, 2012: Poxviridae. In: Virus taxonomy: classification and nomenclature of viruses: Ninth Report of the International Committee on Taxonomy of Viruses. King AMQ, Adams MJ, Carstens EB, Lefkowitz EJ (Eds.), Elsevier Academic Press, San Diego, pp. 291-309.
Wilson IG, 1997: Inhibition and facilitation of nucleic acid amplification. Appl Environ Microbiol, 63(10), 3741.
Xin Z, Velten JP, Oliver MJ, Burke JJ, 2003: High-throughput DNA extraction method suitable for PCR. Biotechniques, 34(4), 820-827.
Zimmer C, 2015: A planet of viruses. University of Chicago Press, pp. 1-30.