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

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.

[1]  Satoshi Tsuneda,et al.  Real-time quantitative LAMP (loop-mediated isothermal amplification of DNA) as a simple method for monitoring ammonia-oxidizing bacteria. , 2006, Journal of biotechnology.

[2]  Stefano Buono,et al.  Silicon photomultipliers and their bio-medical applications , 2007 .

[3]  Hugh A. Bruck,et al.  Carbon nanotubes with enhanced chemiluminescence immunoassay for CCD-based detection of Staphylococcal enterotoxin B in food. , 2008, Analytical chemistry.

[4]  Y. Kostov.  Photodiode-based detection system for biosensors. , 2009, Methods in molecular biology.

[5]  Surveillance for foodborne disease outbreaks - United States, 2006. , 2009, MMWR. Morbidity and mortality weekly report.

[6]  Hans Lehrach,et al.  Quantitative PCR based expression analysis on a nanoliter scale using polymer nano-well chips , 2007, Biomedical microdevices.

[7]  A. Wolff,et al.  Comparison of multiple DNA dyes for real-time PCR: effects of dye concentration and sequence composition on DNA amplification and melting temperature , 2007, Nucleic acids research.

[8]  Jan Berka,et al.  A massively parallel PicoTiterPlate™ based platform for discrete picoliter‐scale polymerase chain reactions , 2003, Electrophoresis.

[9]  N. Ashbolt,et al.  Environmental microbiology : current technology and water applications , 2010 .

[10]  Michael G. Mauk,et al.  A disposable, integrated loop-mediated isothermal amplification cassette with thermally actuated valves , 2011, Microfluidics and nanofluidics.

[11]  Hui Chen,et al.  Predicting viruses accurately by a multiplex microfluidic loop-mediated isothermal amplification chip. , 2011, Analytical chemistry.

[12]  Chii-Wann Lin,et al.  Compact optical diagnostic device for isothermal nucleic acids amplification , 2008, Sensors and Actuators B: Chemical.

[13]  Takashi Kawana,et al.  Tolerance of loop-mediated isothermal amplification to a culture medium and biological substances. , 2007, Journal of biochemical and biophysical methods.

[14]  Kiyoshi Inoue,et al.  Development and evaluation of a loop-mediated isothermal amplification assay for rapid and simple detection of Campylobacter jejuni and Campylobacter coli. , 2008, Journal of medical microbiology.

[15]  P. Rochelle,et al.  Prioritizing pathogens for potential future regulation in drinking water. , 2009, Environmental science & technology.

[16]  J. Kong,et al.  Loop-mediated isothermal amplification integrated on microfluidic chips for point-of-care quantitative detection of pathogens. , 2010, Analytical chemistry.

[17]  Olaf Piepenburg,et al.  DNA Detection Using Recombination Proteins , 2006, PLoS biology.

[18]  A. Nisler,et al.  Surveillance for foodborne disease outbreaks --- United States, 2007. , 2010, MMWR. Morbidity and mortality weekly report.

[19]  K. Heuner,et al.  Molecular characterization of Legionella pneumophila-induced interleukin-8 expression in T cells , 2010, BMC Microbiology.

[20]  A. Tárnok.  SYTO dyes and histoproteins—Myriad of applications , 2008, Cytometry. Part A : the journal of the International Society for Analytical Cytology.

[21]  Tasuku Yotoriyama,et al.  Point-of-care testing system enabling 30 min detection of influenza genes. , 2011, Lab on a chip.

[22]  Michael J Beach,et al.  HEALTHIER • PEOPLE TM Surveillance for Waterborne-Disease Outbreaks — United States , 1999 – 2000 , 2002 .

[23]  Gunther F. Craun,et al.  Causes of Outbreaks Associated with Drinking Water in the United States from 1971 to 2006 , 2010, Clinical Microbiology Reviews.

[24]  Angelika Niemz,et al.  Point-of-care nucleic acid testing for infectious diseases. , 2011, Trends in biotechnology.

[25]  Carsten O. Daub,et al.  Optimization of turn-back primers in isothermal amplification , 2011, 2011 IEEE International Conference on Bioinformatics and Biomedicine Workshops (BIBMW).

[26]  Yan Xu,et al.  Helicase‐dependent isothermal DNA amplification , 2004, EMBO reports.

[27]  J. Vaquero,et al.  Intercalating Cyanine Dyes for Nucleic Acid Detection , 2009 .

[28]  Yi Guan,et al.  Sensitive and inexpensive molecular test for falciparum malaria: detecting Plasmodium falciparum DNA directly from heat-treated blood by loop-mediated isothermal amplification. , 2006, Clinical chemistry.

[29]  N. Bean,et al.  Surveillance for foodborne-disease outbreaks--United States, 1993-1997. , 2000, MMWR. CDC surveillance summaries : Morbidity and mortality weekly report. CDC surveillance summaries.

[30]  Yasutaka Morita,et al.  Application of a microchamber array for DNA amplification using a novel dispensing method. , 2002, Archives of histology and cytology.

[31]  B. Ge,et al.  Development of a toxR-based loop-mediated isothermal amplification assay for detecting Vibrio parahaemolyticus , 2010, BMC Microbiology.

[32]  L. Jian,et al.  Loop-mediated isothermal amplification establishment for detection of pseudorabies virus , 2008, Journal of Virological Methods.

[33]  P. Yager,et al.  Point-of-care diagnostics for global health. , 2008, Annual review of biomedical engineering.

[34]  T. Notomi,et al.  Loop-mediated isothermal amplification of DNA. , 2000, Nucleic acids research.

[35]  Y. Mori,et al.  Loop-mediated isothermal amplification (LAMP): a rapid, accurate, and cost-effective diagnostic method for infectious diseases , 2009, Journal of Infection and Chemotherapy.

[36]  Y. Mori,et al.  Detection of loop-mediated isothermal amplification reaction by turbidity derived from magnesium pyrophosphate formation. , 2001, Biochemical and biophysical research communications.

[37]  Hiroyuki Fujita,et al.  Loop-mediated isothermal amplification of a single DNA molecule in polyacrylamide gel-based microchamber , 2008, Biomedical microdevices.

[38]  Collin Tranter,et al.  Glass-composite prototyping for flow PCR with in situ DNA analysis , 2010, Biomedical microdevices.