On-line cell lysis of bacteria and its spores using a microfluidic biochip
| Autor: | Marianna Cíchová, Miloslava Prokšová, Hunor Santha, Viktor Mayer, Lívia Tóthová |
|---|---|
| Jazyk: | angličtina |
| Rok vydání: | 2012 |
| Předmět: |
microfluidic biochip
Chromatography Lysis General Immunology and Microbiology QH301-705.5 General Neuroscience sybr green real-time pcr Microfluidics waterborne pathogens Biology biology.organism_classification Molecular biology DNA extraction General Biochemistry Genetics and Molecular Biology genomic DNA chemistry.chemical_compound Chelex 100 chemistry chelex 100 Biology (General) General Agricultural and Biological Sciences Biochip cell lysis DNA Bacteria |
| Zdroj: | Open Life Sciences, Vol 7, Iss 2, Pp 230-240 (2012) |
| ISSN: | 2391-5412 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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