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A CCD-based fluorescence imaging system for real-time loop-mediated isothermal amplification-based rapid and sensitive detection of waterborne pathogens on microchips

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Abstract

Rapid, sensitive, and low-cost pathogen diagnostic systems are needed for early disease diagnosis and treatment, especially in resource-limited settings. This study reports a low-cost charge-coupled device (CCD)-based fluorescence imaging system for rapid detection of waterborne pathogens by isothermal gene amplification in disposable microchips. Fluorescence imaging capability of this monochromatic CCD camera is evaluated by optimizing the gain, offset, and exposure time. This imaging system is validated for 12 virulence genes of major waterborne pathogens on cyclic olefin polymer (COP) microchips, using SYTO-82 dye and real time fluorescence loop-mediated isothermal amplification referred here as microRTf-LAMP. Signal-to-noise ratio (SNR) and threshold time (Tt) of microRTf-LAMP assays are compared with those from a commercial real-time polymerase chain reaction (PCR) instrument. Applying a CCD exposure of 5 s to 105 starting DNA copies of microRTf-LAMP assays increases the SNR by 8-fold and reduces the Tt by 9.8 min in comparison to a commercial real-time PCR instrument. Additionally, single copy level sensitivity for Campylobacter jejuni 0414 gene is obtained for microRTf-LAMP with a Tt of 19 min, which is half the time of the commercial real-time PCR instrument. Due to the control over the exposure time and the wide field imaging capability of CCD, this low-cost fluorescence imaging system has the potential for rapid and parallel detection of pathogenic microorganisms in high throughput microfluidic chips.

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References

  • T. Abe, Y. Segawa, H. Watanabe, T. Yotoriyama, S. Kai, A. Yasuda, N. Shimizu, N. Tojo, Point-of-care testing system enabling 30 min detection of influenza genes. Lab Chip 11, 1166–1167 (2011)

    Article  Google Scholar 

  • Y. Aoi, M. Hosogai, S. Tsuneda, Real-time quantitative LAMP (loop-mediated isothermal amplification of DNA) as a simple method for monitoring ammonia-oxidizing bacteria. J. Biotechnol. 125(4), 484–491 (2006)

    Article  Google Scholar 

  • A. Boore, K.M. Herman, A.S. Perez, C.C. Chen, D.J. Cole, B.E. Mahon, P.M. Griffin, I.T. Williams, A.J. Hall, Surveillance for foodborne disease outbreaks-United States 2007. Morb. Mortal. Wkly. Rep. 59(31), 973–1007 (2010)

    Google Scholar 

  • S. Chen, B. Ge, Development of a toxR-based loop-mediated isothermal amplification assay for detecting Vibrio parahaemolyticus. BMC Microbiol. 10, 1–9 (2010)

    Article  Google Scholar 

  • G.F. Craun, J.M. Brunkard, J.S. Yoder, V.A. Roberts, J. Carpenter, T. Wade, R.L. Calderon, J.M. Roberts, M.J. Beach, S.L. Roy, Causes of outbreaks associated with drinking water in the United States from 1971 to 2006. Clin. Microbiol. Rev. 23(3), 507–528 (2010)

    Article  Google Scholar 

  • A. Dahl, M. Sultan, A. Jung, R. Schwartz, M. Lange, M. Steinwand, K.J. Livak, H. Lehrach, L. Nyarsik, A massively parallel PicoTiterPlate™ based platform for discrete picoliter-scale polymerase chain reactions. Biomed. Microdevices 9, 307–314 (2007)

    Article  Google Scholar 

  • T.G. Deligeorgiev, S. Kaloyanova, J.J. Vaquera, Intercalating cyanine dyes for nucleic acid detection. Recent Pat. Mater. Sci. 2, 1–26 (2009)

    Article  Google Scholar 

  • F.X. En, X. Wei, L. Jian, C. Qin, Loop-mediated isothermal amplification establishment for detection of pseudorabies virus. J. Virol. Methods 151(1), 35–39 (2008)

    Article  Google Scholar 

  • X. Fang, Y. Liu, J. Kong, X. Jiang, Loop-mediated isothermal amplification integrated on microfluidic chips for point-of-care quantitative detection of pathogens. Anal. Chem. 82(7), 3002–3006 (2010a)

    Article  Google Scholar 

  • X. Fang, H. Chen, S. Yu, X. Jiang, J. Kong, Predicting Viruses Accurately by a Multiplex Microfluidic Loop-Mediated Isothermal Amplification Chip. Anal. Chem. 83, 690–695 (2010b)

    Article  Google Scholar 

  • A. Gottscheber, S. Dech, Automatic detection of space debris with a MEADE telescope. 61st International Astronautical Congress, 1–5 (2010)

  • E. Grigoriev, A. Akindinov, M. Breitenmoser, S. Buono, E. Charbon, C. Niclass, I. Desforges, R. Rocca, Silicon photomultipliers and their bio-medical applications. Nucl. Instrum. Methods Phys. Res. Sect. A 571(1–2), 130–133 (2007)

    Article  Google Scholar 

  • H. Gudnason, M. Dufva, D.D. Bang, A. Wolff, Comparison of multiple DNA dyes for real-time PCR: effects of dye concentration and sequence composition on DNA amplification and melting temperature. Nucleic Acids Res. 35(19), e127 (2007)

    Article  Google Scholar 

  • R. Hoffman, M.M. Marshall, M.C. Gibson, P.A. Rochelle, Prioritizing pathogens for potential future regulation in drinking water. Environ. Sci. Technol. 43(14), 5165–5170 (2009)

    Article  Google Scholar 

  • H. Kaneko, T. Kawana, E. Fukushima, T. Suzutani, Tolerance of loop-mediated isothermal amplification to a culture medium and biological substances. J. Biochem. Biophys. Methods 70(3), 499–501 (2007)

    Article  Google Scholar 

  • Y. Kimura, M. J. L. de Hoon, S. Aoki, Y. Ishizu, Y. Kawai, Y Kogo, C. O. Daub, A. Lezhava, E. Arner, Y. Hayashizaki, Optimization of turn-back primers in isothermal amplification. Nucleic Acids Res., 1–8 (2011)

  • Y. Kostov, Methods in Molecular Biology: Biosensors and Biodetection (Humana Press, 2009), pp. 307–323

  • L. Lam, S. Sakakihara, K. Ishizuka, S. Takeuchi, H.F. Arata, H. Fujita, H. Noji, Loop-mediated isothermal amplification of a single DNA molecule in polyacrylamide gel-based microchamber. Biomed. Microdevices 10(4), 539–546 (2008)

    Article  Google Scholar 

  • S.H. Lee, D.A. Levy, G.F. Craun, M.J. Beach, R.L. Calderon, Surveillance for waterborne-disease outbreaks-united States, 1999–2000. Morb. Mortal. Wkly. Rep. 51, 1–28 (2002)

    Google Scholar 

  • S.Y. Lee, J.G. Huang, T.L. Chuang, J.C. Sheu, Y.K. Chuang, M. Holl, D.R. Meldrum, C.N. Lee, C.W. Lin, Compact optical diagnostic device for isothermal nucleic acids amplification. Sensor. Actuat. B-Chem. 133(2), 493–501 (2008)

    Article  Google Scholar 

  • C. Liu, M. G. Mauk, H. H. Bau, A disposable, integrated loop-mediated isothermal ampification cassette with thermally actuated valves. Microfluid. Nanofluid. 10.1007/s10404-10011-10788-10403 (2011)

  • Y. Matsubara, M. Kobayashi, Y. Morita, E. Tamiya, Application of a microchamber array for DNA amplification using a novel dispensing method. Arch. Histol. Cytol. 65(5), 481–488 (2002)

    Article  Google Scholar 

  • Y. Mori, K. Nagamine, N. Tomita, T. Notomi, Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. Biochem. Biophys. Res. Commun. 289(1), 150–154 (2001)

    Article  Google Scholar 

  • Y. Mori, T. Notomi, Loop-mediated isothermal amplification (LAMP): a rapid, accurate, and cost-effective diagnostic method for infectious diseases. J. Infect. Chemother. 15(2), 62–69 (2009)

    Article  Google Scholar 

  • A. Niemz, T.M. Ferguson, D.S. Boyle, Point-of-care nucleic acid testing for infectious diseases. Trends Biotechnol. 29, 240–250 (2011)

    Article  Google Scholar 

  • T. Notomi, H. Okayama, H. Masubuchi, T. Yonekawa, K. Watanabe, N. Amino, T. Hase, Loop-mediated isothermal amplification of DNA. Nucleic Acids Res. 28(12), e63 (2000)

    Article  Google Scholar 

  • O. Piepenburg, C.H. Williams, D.L. Stemple, N.A. Armes, DNA detection using recombination proteins. PLoS Biol. 4(7), 1115–1121 (2006)

    Article  Google Scholar 

  • I. Pjescic, C. Tranter, P.L. Hindmarsh, N.D. Crews, Glass-composite prototyping for flow PCR with in situ DNA analysis. Biomed. Microdevices 12(2), 333–343 (2010)

    Article  Google Scholar 

  • L.L.M. Poon, B.W.Y. Wong, E.H.T. Ma, K.H. Chan, L.M.C. Chow, W. Abeyewickreme, N. Tangpukdee, K.Y. Yuen, Y. Guan, S. Looareesuwan, J.S.M. Peiris, Sensitive and inexpensive molecular test for falciparum malaria: Detecting Plasmodium falciparum DNA directly from heat-treated blood by loop-mediated isothermal amplification. Clin. Chem. 52(2), 303–306 (2006)

    Article  Google Scholar 

  • G. Seyrig, F. Ahmad, R. D. Stedtfeld, D. M. Tourlousse, S. A. Hashsham, Environmental Microbiology: Current Technology and Water Applications (Caister Academic Press, 2010), pp. 103–125

  • A. Tarnok, SYTO dyes and histoproteins-myriad of applications. Cytometry A 73 A(6), 477–479 (2008)

    Google Scholar 

  • M. Vincent, Y. Xu, H.M. Kong, Helicase-dependent isothermal DNA amplification. EMBO Reports 5(8), 795–800 (2004)

    Article  Google Scholar 

  • P. Yager, G.J. Domingo, J. Gerdes, Point-of-care diagnostics for global health. Annu. Rev. Biomed. Eng. 10, 107–144 (2008)

    Article  Google Scholar 

  • W. Yamazaki, M. Taguchi, M. Ishibashi, M. Kitazato, M. Nukina, N. Misawa, K. Inoue, Development and evaluation of a loop-mediated isothermal amplification assay for rapid and simple detection of Campylobacter jejuni and Campylobacter coli. J. Med. Microbiol. 57(4), 444–451 (2008)

    Article  Google Scholar 

  • M.H. Yang, Y. Kostov, H.A. Bruck, A. Rasooly, Carbon Nanotubes with Enhanced Chemiluminescence Immunoassay for CCD-Based Detection of Staphylococcal Enterotoxin B in Food. Anal. Chem. 80(22), 8532–8537 (2008)

    Article  Google Scholar 

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Acknowledgements

This research has been partially supported by grants from the US Environmental Protection Agency (Grant Number: RD83301001 and EPD10016) and Michigan Economic Development Corporation (Grant Number: 06–1-P1–0557). We thank Tiffany M. Stedtfeld for helping with reagent preparation. We are grateful to Dr. Alison M. Cupples (Department of Civil and Environmental Engineering, Michigan State University) for allowing us to use real-time PCR instrument (Chromo4TM) in her lab.

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Authors and Affiliations

  1. Department of Civil and Environmental Engineering, Michigan State University, A126 Research Complex-Engineering, East Lansing, MI, 48824, USA

    Farhan Ahmad, Gregoire Seyrig, Dieter M. Tourlousse, Robert D. Stedtfeld & Syed A. Hashsham

  2. The Center for Microbial Ecology, Michigan State University, East Lansing, MI, 48824, USA

    James M. Tiedje & Syed A. Hashsham

  3. Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI, 48824, USA

    James M. Tiedje

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  1. Farhan Ahmad
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  2. Gregoire Seyrig
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  3. Dieter M. Tourlousse
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  4. Robert D. Stedtfeld
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  5. James M. Tiedje
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  6. Syed A. Hashsham
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Correspondence to Syed A. Hashsham.

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Ahmad, F., Seyrig, G., Tourlousse, D.M. et al. A CCD-based fluorescence imaging system for real-time loop-mediated isothermal amplification-based rapid and sensitive detection of waterborne pathogens on microchips. Biomed Microdevices 13, 929–937 (2011). https://doi.org/10.1007/s10544-011-9562-2

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