Skip to main content
SpringerLink
Log in

On-line cell lysis of bacteria and its spores using a microfluidic biochip

  • Research Article
  • Published:
Central European Journal of Biology

Abstract

Optimal detection of pathogens by molecular methods in water samples depends on the ability to extract DNA rapidly and efficiently. In this study, an innovative method was developed using a microfluidic biochip, produced by microelectrochemical system technology, and capable of performing online cell lysis and DNA extraction during a continuous flow process. On-chip cell lysis based on chemical/physical methods was performed by employing a sufficient blend of water with the lysing buffer. The efficiency of lysis with microfluidic biochip was compared with thermal lysis in Eppendorf tubes and with two commercial DNA extraction kits: Power Water DNA isolation kit and ForensicGEM Saliva isolation kit in parallel tests. Two lysing buffers containing 1% Triton X-100 or 5% Chelex were assessed for their lysis effectiveness on a microfluidic biochip. SYBR Green real-time PCR analysis revealed that cell lysis on a microfluidic biochip using 5% Chelex buffer provided better or comparable recovery of DNA than commercial isolation kits. The system yielded better results for Gram-positive bacteria than for Gram-negative bacteria and spores of Gram-positive bacteria, within the limits of detection at 103 CFU/ml. During the continuous flow process in the system, rapid cells lysis with PCR-amplifiable genomic DNA were achieved within 20 minutes.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  1. Prüss A., Kay D., Fewtrell L., Bartram J., Estimating the burden of disease from water, sanitation, and hygiene at a global level, Environ. Health Perspect, 2002, 110, 5537–5542

    Article  Google Scholar 

  2. Khan A.S., Swedlow D.L., Juranek D.D., Precautions against biological and chemical terrorism directed at food and water supplies, Public Health Rep., 2001, 116, 3–14

    PubMed  CAS  Google Scholar 

  3. WHO, Guidelines for drinking-water quality, 3rd ed., Recommendations, Geneva, World Health Organization, 2004

    Google Scholar 

  4. Rompré A., Servais P., Baudart J., De-Roubin M.R., Laurent P., Detection and enumeration of coliforms in drinking water: currenr methods and emerging approaches, J Microbiol. Methods, 2002, 49, 31–54

    Article  PubMed  Google Scholar 

  5. Alexandrino M., Grohmann E., Szewzyk U., Optimization of PCR-based methods for rapid detection of Campalobacter jejuni, Campylobacter coli and Yersinia enterocolitica serovar 0:3 in wastewater samples, Water Res., 2004, 38, 1340–1346

    Article  PubMed  CAS  Google Scholar 

  6. Northrup M.A, Ching M.T, White R.M, Watson R.T., DNA amplification with a microfabricated reaction chamber. In: Proceedings of Transducers’ 93, the Seventh International Conference on Solid-State Sensors and Actuators (7–10 June, 1993, Yokohama, Japan), New York Institute of Electrical and Electronic Engineers, 1993, 924–926

    Google Scholar 

  7. Fan Z.H, Mangru S., Granzow R., Heaney P., Ho W., Dong, Q. et al., Dynamic DNA hybridization on a chip using paramagnetic beads, Anal Chem, 1999, 71, 4851–4859

    Article  PubMed  CAS  Google Scholar 

  8. Kwakye S., Goral V.N., Baeumner A.J., Electrochemical microfluidic biosensor for nucleic acid detection with integrated minipotentiostat, Biosens. Bioelectron., 2006, 21, 2217–2223

    Article  PubMed  CAS  Google Scholar 

  9. Soumet C., Ermel G., Fach P., Colin P., Evaluation of different DNA extraction procedures for the detection of Salmonella from chicken products by polymerase chain reaction, Lett. Appl. Microbiol., 1994, 19, 294–298

    Article  PubMed  CAS  Google Scholar 

  10. Goldenberger D., Perschil I., Ritzler, M. Altwegg, M., A simple ‘universal’ DNA extraction procedure using SDS and proteinase K is compatible with direct DNA amplification PCR, Genome Res., 1995, 4, 368–370

    Article  CAS  Google Scholar 

  11. Taylor M.T., Belgrader P., Furman B.J., Pourahmadi F., Kovacs G.T.A., Northrup, M.A., Lysing bacterial spores by sonication through a flexible interface in a microfluidic system, Anal. Chem., 2001, 73, 492–496

    Article  PubMed  CAS  Google Scholar 

  12. Keshavaraz-Moore E., Hoare M., Dunnill P., Disruption of Baker’syeast in a high-pressure homogenizer: New evidence on mechanism, Enzyme Microb. Technol., 1990, 12, 764–770

    Article  Google Scholar 

  13. Han F., Wang Y., Sims C.E., Bachman M., Chang R., et al., Fast electrical lysis of cells for capillary electrophoresis, Anal. Chem., 2003, 75, 3688–3696

    Article  PubMed  CAS  Google Scholar 

  14. Gao J., Yin X.F., Fang Z.L., Integration of single cell injections, cell lysis, separation and detectionof intracellular constituents on a microfluidic chip, Lab Chip, 2004, 4, 24–52

    Article  Google Scholar 

  15. Xing Ch., Da-fu C., Changchun L., On-line cell lysis and DNA extraction on a microfluidic biochip fabricated by microelectromechanical system technology, Electrophoresis, 2008, 29, 1844–1851

    Article  Google Scholar 

  16. Sethu P., Anahtar M., Moldawer L.L., Continuous row microfluidic device for rapid erythrocyte lysis, Anal. Chem., 2004, 76, 6247–6253

    Article  PubMed  CAS  Google Scholar 

  17. Mahalanabis M., Al-Muayad H., Kulinski D., Altman D., Klapperich C.M., Cell lysis and DNA extraction of gram-positive and gram-negative bacteria from whole blood in a disposable microfluidic chip, Lab Chip, 2009, 9, 2811–2817

    Article  PubMed  CAS  Google Scholar 

  18. Lee J.G, Cheong K.H., Huh N., Kim S., Choi J.W., Ko, C.H., Microchip-based one step DNA extraction and real-time PCR in one chamber for rapid pathogen identification, Lab Chip, 2006, 5, 886–895

    Article  Google Scholar 

  19. Rawsthorne H., Dock C.N., Jaykus L.A., PCR-based method using propidium monoazide to distinguish viable from nonviable Bacillus subtilis spores, Appl. Environ. Microbiol., 2009, 75, 2936–2939

    Article  PubMed  CAS  Google Scholar 

  20. Werners K., Heuvelman C.J., Chakraborty T., Notermans S.H.W., Use of the polymerase chain reaction for direct detection of Listeria monocytogenes in soft cheese, J. Appl. Bacteriol., 1991, 70, 121–126

    Article  Google Scholar 

  21. Khan I.U., Yaday J.S., Development of a singletube, cell lysis-based, genus-specific PCR method for rapid identification of mycobacteria, optimization of cell lysis, PCR primers and conditions and restriction pattern analysis, J. Clin. Microbiol., 2004, 42, 453–457

    Article  PubMed  CAS  Google Scholar 

  22. Suenaga E., Nakamura H., Evaluation of three methods for effective extraction of DNA from human hair, J. Chromatogr. B., 2005, 820, 137–141

    Article  CAS  Google Scholar 

  23. Drahovska H., Turna J., Piknova E., Kuchta T., Szitasova, I. et al., Detection of Salmonella by polymerase chain reaction targeted to fimC gene, Biologia, 2001, 56, 611–616

    CAS  Google Scholar 

  24. Ke D., Picard F.J., Martineau F., Ménard C.H., Roy P.H., Development of a PCR assay for rapid detection of enterococci, J. Clin. Microbiol., 1999, 37, 3497–3503

    PubMed  CAS  Google Scholar 

  25. Aldous W.K., Pounder J.I., Cloud J.L., Woods G.L., Comparison of six methods of extracting Mycobaterium tuberculosis DNA from processed sputum for testing by quantitative real-time PCR, J.Clin. Microbiol., 2005, 43, 2471–2473

    Article  PubMed  CAS  Google Scholar 

  26. Gonzalez Garcia L.A., Rodrigo Tapia J.P., Sanchez L.P., Ramos S., Suarez N.C., DNA extraction using chelex resin for the oncogenic amplification analysis in head and neck tumours, Acta Otorinolaringol. Esp., 2004, 55, 139–144

    Google Scholar 

  27. Aranishi F., Okimoto T., A simple and reliable method for DNA extraction from bivalve mantle, J. Appl. Genet., 2006, 47, 251–254

    Article  PubMed  Google Scholar 

  28. Desloire S., Valiente Moro, C., Chauve, C., Zenner, L., Comparison of four methods of extracting DNA from D. Gallinae, Vet. Res., 2006, 25, 725–732

    Article  Google Scholar 

  29. Burns M.A., Johnson B.N., Brahmasandra S.N., Handique K., An integrated nanoliter analysis device, Science, 1998, 282, 484–487

    Article  PubMed  CAS  Google Scholar 

  30. Karle M., Miwa J., Czilwik G., Auwärter V., Roth G., Zengerle R,. Von Stetten F., Continuous microfluidic DNA extraction using phase-transfer magnetophoresis, Lab Chip, 2010, 10, 3284–3290

    Article  PubMed  CAS  Google Scholar 

  31. Carlo D.D, Jeong K.H, Lee L.P, Reagentless mechanical cell lysis by nanoscale barbs in microchannels for sample preparation, Lab Chip, 2003, 3, 287–291

    Article  PubMed  Google Scholar 

  32. Waters L.C., Jacobson S.C., Kroutchinina N., Khandurina J., Foote R.S., Ramsey J.M., Multiplex sample PCR amplification and electrophoretic analysis on a microchip, Anal. Chem, 1998, 70, 5172–5176

    Article  PubMed  CAS  Google Scholar 

  33. Lee S.W., Tai I.C., Micro cell lysis device, Sensor. Actuat. A-Phys., 1999, 73, 74–79

    Article  Google Scholar 

  34. LaMontagne M.G., Michel F.C., Jr. Holden P.A., Reddy C.A., Evaluation of extraction and purification methods for obtaining PCRamplifiable DNA from compost for microbial community Analysis, J Microbiol Methods, 2002, 49, 255–264

    Article  PubMed  CAS  Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Water Research Institute, National Water Reference Laboratory in Slovakia, 812 49, Bratislava, Slovak Republic

    Marianna Cíchová, Miloslava Prokšová & Lívia Tóthová

  2. Department of Electronics Technology, Budapest University of Technology and Economics, H-1111, Budapest, Hungary

    Hunor Sántha & Viktor Mayer

Authors
  1. Marianna Cíchová
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Miloslava Prokšová
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Lívia Tóthová
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Hunor Sántha
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Viktor Mayer
    View author publications

    You can also search for this author in PubMed Google Scholar

About this article

Cite this article

Cíchová, M., Prokšová, M., Tóthová, L. et al. On-line cell lysis of bacteria and its spores using a microfluidic biochip. cent.eur.j.biol. 7, 230–240 (2012). https://doi.org/10.2478/s11535-012-0005-8

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.2478/s11535-012-0005-8

Keywords

Search

Navigation