Comparison of DNA Extraction Methods for Molecular Detection of Probiotic Lactobacilli, Lysis-resistant Bacteria

Document Type : Original Paper


1 Department of Food Biotechnology, Research Institute of Food Science and Technology, Mashhad, Iran

2 Department of Anesthesiology and Critical Care, Mashhad University of Medical Sciences, Mashhad, Iran


DNA extraction is a crucial step in all nucleic acid-based protocols to identify microorganisms. Lactic acid bacteria are a significant part of healthy microbiota in the human gastrointestinal tract. These gram-positive bacteria have several layers of peptidoglycan in the cell walls. These structures cause difficulties in the cell lysis and obtaining reliable protocols for DNA isolations. The purpose of this study was to assess the autoclave and lysozyme-based DNA purification approaches for achieving the high-quality genomic DNAs of Lactobacillus acidophilus bacteria. DNA concentrations and qualities were also compared with the commercial kit. The results showed that the proper DNA isolation methods were various, according to the downstream applications. Protocols that included lysozyme produced a higher amount of DNA than the autoclave method. Lysozyme treatment combined with silica
-guanidinethiocyanate procedure was the efficient protocol with affordable cost for routine lysis of L. acidophilus bacteria. Appropriate DNA concentration and quality were obtained through this method comparable to those of the commercial kit. Inversely, autoclave treatment had little effect on the breakage of the cell walls indicating low concentrations of extracted DNAs. This method could not completely break down all the bacterial cell walls. However, the breakage of low numbers of cell walls was microscopically observed in the supernatant of the autoclaved cell suspension. The quality of this protocol was found to be adequate for performing direct polymerase chain reaction (PCR) assay on samples with large amounts of lactobacilli. These conclusions suggest attentively selecting the DNA extraction method based on the planned downstream analysis of PCR products.


Main Subjects

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Alimolaei, M., & Golchin, M. (2016). An Efficient DNA Extraction Method for Lactobacillus casei, a Difficult-to-Lyse Bacterium. International Journal of Enteric Pathogens, 4(1), 7-32472.
Angelescu, I.-R., Zamfir, M., Stancu, M.-M., & Grosu-Tudor, S.-S. (2019). Identification and probiotic properties of lactobacilli isolated from two different fermented beverages. Annals of Microbiology, 69(13), 1557-1565.
Boom, R., Sol, C. J., Salimans, M. M., Jansen, C. L., Wertheim-van Dillen, P. M., & van der Noordaa, J. (1990). Rapid and simple method for purification of nucleic acids. J Clin Microbiol, 28(3), 495-503.
De, S., Kaur, G., Roy, A., Dogra, G., Kaushik, R., Yadav, P., . . . Goswami, S. L. (2010). A Simple Method for the Efficient Isolation of Genomic DNA from Lactobacilli Isolated from Traditional Indian Fermented Milk (dahi). Indian J Microbiol, 50(4), 412-418.
Douglas, C. A., Ivey, K. L., Papanicolas, L. E., Best, K. P., Muhlhausler, B. S., & Rogers, G. B. (2020). DNA extraction approaches substantially influence the assessment of the human breast milk microbiome. Scientific Reports, 10(1), 123.
Ehsanbakhsh, M., Sadeghi, A., Raeisi, M., Ebrahimi, M., & Kashaninejad, M. (2017). Isolation, Molecular Identification and Evaluation of Antifungal Effect of Dominant Lactic Acid Bacteria Isolated from Wheat Bran and Rice Bran Sourdoughs. Research and Innovation in Food Science and Technology, 6(3), 245-260.
Harrel, M., & Holmes, A. S. (2022). Review of direct PCR and Rapid DNA approaches to streamline sexual assault kit testing. J Forensic Sci, 67(4), 1336-1347.
Ketchum, R. N., Smith, E. G., Vaughan, G. O., Phippen, B. L., McParland, D., Al-Mansoori, N., . . . Reitzel, A. M. (2018). DNA Extraction Method Plays a Significant Role When Defining Bacterial Community Composition in the Marine Invertebrate Echinometra mathaei [Original Research]. Frontiers in Marine Science, 5.
Lim, M. Y., Song, E. J., Kim, S. H., Lee, J., & Nam, Y. D. (2018). Comparison of DNA extraction methods for human gut microbial community profiling. Syst Appl Microbiol, 41(2), 151-157.
Markowiak, P., & Śliżewska, K. (2017). Effects of Probiotics, Prebiotics, and Synbiotics on Human Health. Nutrients, 9(9).
Quigley, L., O'Sullivan, O., Beresford, T. P., Paul Ross, R., Fitzgerald, G. F., & Cotter, P. D. (2012). A comparison of methods used to extract bacterial DNA from raw milk and raw milk cheese. J Appl Microbiol, 113(1), 96-105.
Shakeri, M.-S., Shahidi, F., Mortazavi, A., Bahrami, A. R., & Nassiri, M. R. (2014). Application of PCR Technique in Combination with DNase Treatment for Detection of Viable Lactobacillus acidophilus Bacteria. Journal of Food Quality, 37(4), 291-295.
Shakeri, M.-S., Shahidi, F., Mortazavi, A., Bahrami, A. R., & Nassiri, M. R. (2018). Combination of competitive PCR and cultivation methods for differential enumeration of viable Lactobacillus acidophilus in bio-yoghurts. International Journal of Dairy Technology, 71(4), 887-892.
Shehadul Islam, M., Aryasomayajula, A., & Selvaganapathy, P. R. (2017). A Review on Macroscale and Microscale Cell Lysis Methods. Micromachines, 8(3), 83.
Simmon, K. E., Steadman, D. D., Durkin, S., Baldwin, A., Jeffrey, W. H., Sheridan, P., . . . Shields, M. S. (2004). Autoclave method for rapid preparation of bacterial PCR-template DNA. J Microbiol Methods, 56(2), 143-149.
Urbaniak, J., Janowski, D., & Jacewski, B. (2019). Isolation of nucleic acids using silicon dioxide powder as a tool for environmental monitoring. Environmental Monitoring and Assessment, 191(12), 732.
Vermassen, A., Leroy, S., Talon, R., Provot, C., Popowska, M., & Desvaux, M. (2019). Cell Wall Hydrolases in Bacteria: Insight on the Diversity of Cell Wall Amidases, Glycosidases and Peptidases Toward Peptidoglycan [Review]. Frontiers in Microbiology, 10.
Videvall, E., Strandh, M., Engelbrecht, A., Cloete, S., & Cornwallis, C. K. (2017). Direct PCR Offers a Fast and Reliable Alternative to Conventional DNA Isolation Methods for Gut Microbiomes. mSystems, 2(6).
Yoo, J. H. (2018). Review of Disinfection and Sterilization - Back to the Basics. Infection & chemotherapy, 50(2), 101-109.
Yuan, S., Cohen, D. B., Ravel, J., Abdo, Z., & Forney, L. J. (2012). Evaluation of Methods for the Extraction and Purification of DNA from the Human Microbiome. PLOS ONE, 7(3), e33865.
Volume 11, Issue 4
February 2023
Pages 415-422
  • Receive Date: 08 May 2022
  • Revise Date: 20 August 2022
  • Accept Date: 23 August 2022