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Journals / Hacettepe Journal of Biology and Chemistry / 2020 / Cilt: 48 - Sayı: 1

Comparison of Bacterial DNA Extraction Methods From Stool Samples for Quantitative Real-Time PCR Analysis

Dışkı Örneklerinde Real-Time PCR Analizleri için Bakteriyel DNA Izolasyon Yöntemlerinin Karşılaştırılması

Ceren ÖZKUL
Pages
41–48
Publication Date
2022-10-17
DOI
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AI Summary & Key Takeaways

AI Insight
⚡ TL;DR(Too Long; Didn't Read)

Commercial kits have better results in terms of DNA recovery and purity, andhave more accurate results for qPCR experiments.

📌 3 Key Takeaways

🎯
Objective & Hypothesis:The most crucial step in molecular microbiology studies is choosing an appropriate procedure to obtain sufficient, highquality DNA.
📊
Key Findings & Data:DNA yield was significantly higher in Invitek when compared to other methods.
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Conclusion & Impact:Commercial kits have better results in terms of DNA recovery and purity, andhave more accurate results for qPCR experiments.
🔬Design:Research Article
📍Scope:Comprehensive Sample
⚙️Variables:Domain Parameters

Abstract

The most crucial step in molecular microbiology studies is choosing an appropriate procedure to obtain sufficient, highquality DNA. Two commercially available kits (QIAamp DNA Stool Mini Kit, Qiagen and PSP Spin Stool DNA Plus Kit, Invitek)and a modified in-house phenol-chloroform extraction method was compared for their efficiency in isolation of certainbacteria from human stool samples for Real-Time qPCR. DNA yield was significantly higher in Invitek when compared to other methods. Spiking experiments and qPCR results revealed that efficiency was in phenol-chloroform extraction was lowerwhen compared to other two commercial kits. Commercial kits have better results in terms of DNA recovery and purity, andhave more accurate results for qPCR experiments.

Özet

Moleküler mikrobiyoloji çalışmalarında en önemli basamak yüksek kalitede yeterli DNA eldesidir. Bu çalışmada iki hazır ticari izolasyon kiti (QIAamp DNA Stool Mini Kit, Qiagen and PSP Spin Stool DNA Plus Kit, Inivtek) ve modifiye fenol-kloroform ekstraksiyon yöntemi Real-Time qPCR analizleri için belirli bakterilerin dışkıdan izolasyonundaki etkinleri bakımından karşılaştırılmıştır. DNA miktarı Invitek kitinde en yüksek oranda elde edilmiştir. Dışkıya ve PCR karışımına bilinen miktarda hücre ve DNA eklenme deneyleri ve qPCR sonuçları fenol-kloroform ekstraksiyon yönteminin etkinliğinin düşük olduğunu göstermiştir. Hazır ticari dışkıdan izolasyon kitleri DNA eldesi ve saflığı açsıından qPCR deneylerinde kullanılmak üzere daha doğru sonuçlar vermektedir.

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Cite This Article

ÖZKUL, C. (2022). Comparison of Bacterial DNA Extraction Methods From Stool Samples for Quantitative Real-Time PCR Analysis. Hacettepe Journal of Biology and Chemistry, Cilt: 48 - Sayı: 1, 41–48. https://ajindex.com/articles/528974

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References (14)

  1. P.J. Turnbaugh, R.E. Ley, M. Hamady, C.M. Fraser-Liggett, R. Knight, J.I. Gordon, The human microbiome project, Nature, 449 (2007) 804-810
  2. F. Guarner, J.R. Malagelada, Gut flora in health and disease, Lancet, 361 (2003) 512-519
  3. M.C. Abt, D. Artis, The intestinal microbiota in health and disease: the influence of microbial products on immune cell homeostasis, Curr. Opin. Gastroenterol., 25 (2009) 496-502
  4. N.A. Bokulich, J. Chung, T. Battaglia, N. Henderson, M. Jay, H. Li, A. D. Lieber, et al., Antibiotics, birth mode, and diet shape microbiome maturation during early life, Sci. Transl. Med., 8 (2016) 343ra382
  5. P.J. Turnbaugh, M. Hamady, T. Yatsunenko, B.L. Cantarel, A. Duncan, R.E. Ley, M.L. Sogin, et al., A core gut microbiome in obese and lean twins, Nature, 457 (2009) 480-484
  6. P.J. Turnbaugh, R.E. Ley, M.A. Mahowald, V. Magrini, E.R. Mardis, J.I. Gordon, An obesity-associated gut microbiome with increased capacity for energy harvest, Nature, 444 (2006) 1027-1031
  7. G.M. Weinstock, Genomic approaches to studying the human microbiota, Nature, 489 (2012) 250-256
  8. E.R. Mardis, Next-generation DNA sequencing methods, Annu. Rev. Genomics. Hum. Genet., 9 (2008) 387-402
  9. R.E. Ley, F. Backhed, P. Turnbaugh, C.A. Lozupone, R.D. Knight, J.I. Gordon, Obesity alters gut microbial ecology, Proc. Natl. Acad. Sci. U S A., 102 (2005) 11070-11075
  10. I. Sekirov, S.L. Russell, L.C. Antunes, B.B. Finlay, Gut microbiota in health and disease, Physiol. Rev., 90 (2010) 859-904
  11. R. Poretsky, R.L. Rodriguez, C. Luo, D. Tsementzi, K.T. Konstantinidis, Strengths and limitations of 16S rRNA gene amplicon sequencing in revealing temporal microbial community dynamics, PLoS One, 9 (2014) e93827
  12. A. Rintala, S. Pietila, E. Munukka, E. Eerola, J.P. Pursiheimo, A. Laiho, S. Pekkala, et al., Gut Microbiota Analysis Results Are Highly Dependent on the 16S rRNA Gene Target Region, Whereas the Impact of DNA Extraction Is Minor, J. Biomol. Tech., 28 (2017) 19-30
  13. X. Guo, X. Xia, R. Tang, J. Zhou, H. Zhao, K. Wang, Development of a real-time PCR method for Firmicutes and Bacteroidetes in faeces and its application to quantify intestinal population of obese and lean pigs, Lett. Appl. Microbiol., 47 (2008) 367-373
  14. S.J. Ott, M. Musfeldt, U. Ullmann, J. Hampe, S. Schreiber, Quantification of intestinal bacterial populations by realtime PCR with a universal primer set and minor groove binder probes: a global approach to the enteric flora, J. Clin. Micro