title: A CCD-based fluorescence imaging system for real-time loop-mediated isothermal amplification-based rapid and sensitive detection of waterborne pathogens on microchips.
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 microRT(f)-LAMP. Signal-to-noise ratio (SNR) and threshold time (Tt) of microRT(f)-LAMP assays are compared with those from a commercial real-time polymerase chain reaction (PCR) instrument. Applying a CCD exposure of 5 s to 10(5) starting DNA copies of microRT(f)-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 microRT(f)-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.
authors: Department of Civil and Environmental Engineering, Michigan State University, A126 Research Complex-Engineering, East Lansing, MI 48824, USA.
authors: Farhan
authors: F
authors: Ahmad
authors: Gregoire
authors: G
authors: Seyrig
authors: Dieter M
authors: DM
authors: Tourlousse
authors: Robert D
authors: RD
authors: Stedtfeld
authors: James M
authors: JM
authors: Tiedje
authors: Syed A
authors: SA
authors: Hashsham
mesh_terms: D016123:Campylobacter jejuni / Q000235:genetics / Q000472:pathogenicity*; D004867:Equipment Design; D005453:Fluorescence; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation; D021141:Nucleic Acid Amplification Techniques / Q000295:instrumentation*; D060888:Real-Time Polymerase Chain Reaction; D012680:Sensitivity and Specificity; D059629:Signal-To-Noise Ratio; D014774:Virulence / Q000235:genetics; D014871:Water Microbiology