On-line cell lysis of bacteria and its spores using a microfluidic biochip
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- 作者：Marianna Cíchová (1)
Miloslava Prok?ová (1)
Lívia Tóthová (1)
Hunor Sántha (2)
Viktor Mayer (2) - 关键词：Cell lysis ; Chelex 100 ; Microfluidic biochip ; SYBR Green real ; time PCR ; Waterborne pathogens
- 刊名：Central European Journal of Biology
- 出版年：2012
- 出版时间：April 2012
- 年：2012
- 卷：7
- 期：2
- 页码：230-240
- 全文大小：938KB
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Miloslava Prok?ová (1)
Lívia Tóthová (1)
Hunor Sántha (2)
Viktor Mayer (2)
1. Water Research Institute, National Water Reference Laboratory in Slovakia, 812 49, Bratislava, Slovak Republic
2. Department of Electronics Technology, Budapest University of Technology and Economics, H-1111, Budapest, Hungary - ISSN：1644-3632
文摘
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.