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Making the best of a bad sample: Comparison of DNA extraction and quantification methods using sub-optimally stored Ixodes ricinus ticks.

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  • Additional Information
    • Source:
      Publisher: Public Library of Science Country of Publication: United States NLM ID: 101285081 Publication Model: eCollection Cited Medium: Internet ISSN: 1932-6203 (Electronic) Linking ISSN: 19326203 NLM ISO Abbreviation: PLoS One Subsets: MEDLINE
    • Publication Information:
      Original Publication: San Francisco, CA : Public Library of Science
    • Subject Terms:
      Ixodes*/genetics ; Ixodes*/microbiology ; DNA*/isolation & purification ; Specimen Handling*/methods; Animals ; Male ; Female ; Norway ; DNA, Bacterial/isolation & purification
    • Abstract:
      Competing Interests: The authors have declared that no competing interests exist.
      The primary aim of this study was to evaluate the performance of four DNA extraction methods on a collection of 200 sub-optimally stored Ixodes ricinus ticks from Southeastern Norway (177 nymphs, 11 males and 12 females). The methods were ammoniium hydroxide hydrolysis of homogenised or intact ticks and two commercial silica membrane-based kits, the QIAGEN Blood and Tissue kit and the QIAGEN Mini kit. DNA was evaluated by spectrophotometry, fluorometry, agarose gel electrophoresis and quantitative PCR. The second aim was to compare methods of evaluating the yield and purity. All four extraction methods provided amplifiable DNA, but the yield was variable (median 151 ng, range 0.13 to 4500 ng). DNA yields were not significantly different across the methods. Nine of 200 samples were inhibitory, all of which were ammonium hydroxide extracts of homogenised ticks. DNA purity, as judged by A260/280 ratios, was low; it was highest (mean = 1.63) for the Qiagen Blood and Tissue kit with the other methods showing values averaging 1.44. DNA yield measurements using qPCR, fluorometry (Qubit), drop spectrophotometry (NanoDrop) and gel electrophoresis (E-gels) correlated poorly, r ranging from <0 to 0.9 with no systematic pattern (average 0.4), probably reflecting the effects of low purity, low concentrations and differing amounts of single- and double-stranded DNA. Neoehrlichia mikurensis and Borrelia burgdorferi s.l. were detected in 5 and 13% of samples, respectively by qPCR. Our findings indicate that for the purposes of qPCR analysis, ammoniium hydroxide hydrolysis of intact ticks, a very cheap and simple method is as good as any of the other methods tested.
      (Copyright: © 2025 Cotes-Perdomo et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)
    • References:
      Clin Microbiol Rev. 2006 Jan;19(1):165-256. (PMID: 16418529)
      Asian J Transfus Sci. 2019 Jul-Dec;13(2):95-99. (PMID: 31896914)
      Exp Appl Acarol. 1995 Aug;19(8):473-8. (PMID: 8529479)
      BMC Biotechnol. 2003 Oct 28;3:20. (PMID: 14583097)
      PeerJ. 2015 Aug 13;3:e1147. (PMID: 26290800)
      PeerJ. 2021 Feb 12;9:e10799. (PMID: 33614282)
      Ticks Tick Borne Dis. 2019 Aug;10(5):1041-1045. (PMID: 31171466)
      BMC Microbiol. 2019 Aug 28;19(1):199. (PMID: 31462211)
      Ticks Tick Borne Dis. 2017 Jan;8(1):185-189. (PMID: 27825733)
      Clin Microbiol Infect. 2009 Dec;15 Suppl 2:43-5. (PMID: 20584162)
      PLoS One. 2016 Mar 03;11(3):e0150528. (PMID: 26937682)
      Anal Bioanal Chem. 2018 Apr;410(10):2569-2583. (PMID: 29504082)
      Ticks Tick Borne Dis. 2017 Feb;8(2):235-240. (PMID: 27856176)
      J Mol Biol. 1962 Jun;4:467-87. (PMID: 13897515)
      PLoS One. 2013 Jun 06;8(6):e62692. (PMID: 23762227)
      Parasitology. 2004;129 Suppl:S3-14. (PMID: 15938502)
      J Clin Microbiol. 2014 Jun;52(6):2139-43. (PMID: 24740078)
      J Forensic Sci. 2014 Nov;59(6):1517-29. (PMID: 25182468)
      Parasit Vectors. 2018 Jan 4;11(1):12. (PMID: 29301588)
      Exp Appl Acarol. 1999 Dec;23(12):929-60. (PMID: 10737729)
      J Clin Pathol. 1991 Jul;44(7):610-1. (PMID: 1856296)
      Mol Ecol Resour. 2017 Jul;17(4):686-693. (PMID: 27768249)
      J Bacteriol. 1976 Sep;127(3):1550-7. (PMID: 8432)
      Med Vet Entomol. 2011 Mar;25(1):109-12. (PMID: 20704654)
      Eur J Clin Microbiol Infect Dis. 2024 Jul;43(7):1261-1295. (PMID: 38676855)
      Sci Rep. 2016 Apr 06;6:24067. (PMID: 27048884)
      J Parasitol. 2000 Feb;86(1):38-43. (PMID: 10701561)
      Vector Borne Zoonotic Dis. 2007 Winter;7(4):555-61. (PMID: 17979538)
      Acta Vet Scand. 2009 Nov 27;51:47. (PMID: 19943915)
      Exp Appl Acarol. 2006;38(2-3):189-99. (PMID: 16596352)
      Exp Appl Acarol. 2012 Dec;58(4):431-9. (PMID: 22684812)
      J Clin Microbiol. 2001 Oct;39(10):3666-71. (PMID: 11574588)
      Clin Chim Acta. 2018 Apr;479:14-19. (PMID: 29309771)
      Microorganisms. 2022 Jun 20;10(6):. (PMID: 35744772)
    • Accession Number:
      9007-49-2 (DNA)
      0 (DNA, Bacterial)
    • Publication Date:
      Date Created: 20250529 Date Completed: 20250529 Latest Revision: 20250601
    • Publication Date:
      20250601
    • Accession Number:
      PMC12121741
    • Accession Number:
      10.1371/journal.pone.0323251
    • Accession Number:
      40440307