title: A signal-amplified electrochemical DNA biosensor incorporated with a colorimetric internal control for Vibrio cholerae detection using shelf-ready reagents.
abstract: A novel enzyme/nanoparticle-based DNA biosensing platform with dual colorimetric/electrochemical approach has been developed for the sequence-specific detection of the bacterium Vibrio cholerae, the causative agent of acute diarrheal disease in cholera. This assay platform exploits the use of shelf-stable and ready-to-use (shelf-ready) reagents to greatly simplify the bioanalysis procedures, allowing the assay platform to be more amenable to point-of-care applications. To assure maximum diagnosis reliability, an internal control (IC) capable of providing instant validation of results was incorporated into the assay. The microbial target, single-stranded DNA amplified with asymmetric PCR, was quantitatively detected via electrochemical stripping analysis of gold nanoparticle-loaded latex microspheres as a signal-amplified hybridization tag, while the incorporated IC was analyzed using a simplified horseradish peroxidase enzyme-based colorimetric scheme by simple visual observation of enzymatic color development. The platform showed excellent diagnostic sensitivity and specificity (100%) when challenged with 145 clinical isolate-spiked fecal specimens. The limits of detection were 0.5ng/ml of genomic DNA and 10 colony-forming units (CFU)/ml of bacterial cells with dynamic ranges of 0-100ng/ml (R2=0.992) and log10(1-104CFU/ml) (R2=0.9918), respectively. An accelerated stability test revealed that the assay reagents were stable at temperatures of 4-37°C, with an estimated ambient shelf life of 200 days. The versatility of the biosensing platform makes it easily adaptable for quantitative detection of other microbial pathogens.
authors: Faculty of Applied Sciences, Universiti Teknologi MARA, Tapah Campus, Tapah Road, Perak, 35400 Malaysia. Electronic address: lowkimfatt@perak.uitm.edu.my.
authors: Kim-Fatt
authors: KF
authors: Low
authors: Faculty of Applied Sciences, Universiti Teknologi MARA, Shah Alam, Selangor, 40450 Malaysia.
authors: Zainiharyati Mohd
authors: ZM
authors: Zain
authors: Department of Medical Microbiology and Parasitology, School of Medical Sciences, Universiti Sains Malaysia, Kubang Kerian, Kelantan, 16150 Malaysia. Electronic address: yeancyn@yahoo.com.
authors: Chan Yean
authors: CY
authors: Yean
mesh_terms: D015374:Biosensing Techniques / Q000295:instrumentation / Q000379:methods*; D002771:Cholera / Q000175:diagnosis* / Q000382:microbiology; D003124:Colorimetry / Q000295:instrumentation / Q000379:methods; D004269:DNA, Bacterial / Q000032:analysis / Q000235:genetics; D055664:Electrochemical Techniques / Q000295:instrumentation / Q000379:methods*; D004867:Equipment Design; D005243:Feces / Q000382:microbiology*; D006046:Gold / Q000737:chemistry; D006801:Humans; D053768:Metal Nanoparticles / Q000737:chemistry; D009693:Nucleic Acid Hybridization / Q000379:methods; D019095:Point-of-Care Systems; D015203:Reproducibility of Results; D014734:Vibrio cholerae / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D004269:DNA, Bacterial; D006046:Gold
keywords: Colorimetric detection; DNA biosensor; Electrochemical detection; Internal control; Shelf-ready reagent; Signal amplification