Increasing PCR sensitivity by removal of polymerase inhibitors in environmental samples by using dielectrophoresis

被引:20
作者
del Carmen Jaramillo, Maria [1 ]
Martinez-Duarte, Rodrigo [2 ]
Huettener, Mario [1 ]
Renaud, Philippe [2 ]
Torrents, Eduard [1 ]
Juarez, Antonio [1 ,3 ]
机构
[1] Inst Bioengn Catalonia IBEC, Barcelona 08028, Spain
[2] Ecole Polytech Fed Lausanne, Microsyst Lab LMIS4, CH-1015 Lausanne, Switzerland
[3] Univ Barcelona, Dept Microbiol, Fac Biol, E-08028 Barcelona, Spain
关键词
Yeast; Dielectrophoresis; PCR; Separation; Carbon-electrode; CONTINUOUS SEPARATION; CELLS; MICROORGANISMS; PARTICLES; IMPEDANCE; MIXTURES;
D O I
10.1016/j.bios.2012.12.049
中图分类号
Q6 [生物物理学];
学科分类号
071011 ;
摘要
Dielectrophoresis (DEP) is a powerful tool to manipulate cells and molecules in microfluidic chips. However, few practical applications using DEP exist. An immediate practical application of a carbon-electrode DEP system, in removing PCR inhibitors from a sample, is reported in this work. We use a high throughput carbon-electrode DEP system to trap yeast cells from a natural sample (fermented grape must) and then in situ remove contaminants that interfere with PCR analysis. Retrieval of this enriched and purified yeast population from the DEP system then allows for a significant increase of sensitivity during PCR analysis. Furthermore, the fact that DEP can discriminate between viable and non-viable cells minimizes the number of false positives commonly obtained when using PCR alone. Experimental results provide clear evidence that the carbon-electrode DEP-based sample preparation step can readily and effectively clean environmental samples from natural contaminants and improve PCR sensitivity. (c) 2012 Elsevier B.V. All rights reserved.
引用
收藏
页码:297 / 303
页数:7
相关论文
共 51 条
[31]   Review Article-Dielectrophoresis: Status of the theory, technology, and applications [J].
Pethig, Ronald .
BIOMICROFLUIDICS, 2010, 4 (02)
[32]   Continuous analysis of dye-loaded, single cells on a microfluidic chip [J].
Phillips, K. Scott ;
Lai, Hsuan Hong ;
Johnson, Emily ;
Sims, Christopher E. ;
Allbritton, Nancy L. .
LAB ON A CHIP, 2011, 11 (07) :1333-1341
[33]   Pathogen Quantitation in Complex Matrices: A Multi-Operator Comparison of DNA Extraction Methods with a Novel Assessment of PCR Inhibition [J].
Pontiroli, Alessandra ;
Travis, Emma Rachel ;
Sweeney, Francis Patrick ;
Porter, David ;
Gaze, William Hugo ;
Mason, Sam ;
Hibberd, Victoria ;
Holden, Jennifer ;
Courtenay, Orin ;
Wellington, Elizabeth Margaret Helen .
PLOS ONE, 2011, 6 (03)
[34]   Recent advances in quantitative PCR (qPCR) applications in food microbiology [J].
Postollec, Florence ;
Falentin, Helene ;
Pavan, Sonia ;
Combrisson, Jerome ;
Sohier, Daniele .
FOOD MICROBIOLOGY, 2011, 28 (05) :848-861
[35]  
Radstrom Peter, 2003, Methods Mol Biol, V216, P31
[36]   Detection of cellular responses to toxicants by dielectrophoresis [J].
Ratanachoo, K ;
Gascoyne, PRC ;
Ruchirawat, M .
BIOCHIMICA ET BIOPHYSICA ACTA-BIOMEMBRANES, 2002, 1564 (02) :449-458
[37]   PCR Inhibitor Levels in Concentrates of Biosolid Samples Predicted by a New Method Based on Excitation-Emission Matrix Spectroscopy [J].
Rock, Channah ;
Alum, Absar ;
Abbaszadegan, Morteza .
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2010, 76 (24) :8102-8109
[38]   Innovative methods for soil DNA purification tested in soils with widely differing characteristics [J].
Sagova-Mareckova, Marketa ;
Cermak, Ladislav ;
Novotna, Jitka ;
Plhackova, Kamila ;
Forstova, Jana ;
Kopecky, Jan .
APPLIED AND ENVIRONMENTAL MICROBIOLOGY, 2008, 74 (09) :2902-2907
[39]   Improving qPCR efficiency in environmental samples by selective removal of humic acids with DAX-8 [J].
Schriewer, A. ;
Wehlmann, A. ;
Wuertz, S. .
JOURNAL OF MICROBIOLOGICAL METHODS, 2011, 85 (01) :16-21
[40]   Selective isolation of live/dead cells using contactless dielectrophoresis (cDEP) [J].
Shafiee, Hadi ;
Sano, Michael B. ;
Henslee, Erin A. ;
Caldwell, John L. ;
Davalos, Rafael V. .
LAB ON A CHIP, 2010, 10 (04) :438-445