title: A thermostabilized magnetogenosensing assay for DNA sequence-specific detection and quantification of Vibrio cholerae.
abstract: Vibrio cholerae is a human pathogen that causes mild to severe diarrheal illnesses and has major public health significance. Herein, we present a thermostabilized electrochemical genosensing assay combining the use of magnetic beads as a biorecognition platform and gold nanoparticles as a hybridization tag for the detection and quantification of V. cholerae lolB gene single-stranded asymmetric PCR amplicons as an alternative to the time-consuming classical isolation method. This thermostabilized, pre-mixed, pre-aliquoted and ready-to-use magnetogenosensing assay simplified the procedures and permitted the reaction to be conducted at room temperature. The asymmetric PCR amplicons were hybridized to a magnetic bead-functionalized capture probe and a fluorescein-labeled detection probe followed by tagging with gold nanoparticles. Electrochemical detection of the chemically dissolved gold nanoparticles was performed using the differential pulse anodic stripping voltammetry method. The real-time stability evaluation of thermostabilized assay was found to be stable for at least 180 days at room temperature (25-30°C). The analytical specificity of the assay was 100%, while its analytical sensitivity was linearly related to different concentrations of 200-mer synthetic target, purified genomic DNA, and bacterial culture with a limit of detection (LoD) of 3.9nM, 5pg/µl, and 10(3)CFU/ml, respectively. The clinical applicability of the assay was successfully validated using spiked stool samples with an average current signal-to-cut-off ratio of 10.8. Overall, the precision of the assay via relative standard deviation was <10%, demonstrating its reliability and accuracy.
authors.affiliation: Department of Medical Microbiology and Parasitology, School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia. lowkimfatt@hotmail.my
authors.forename: Kim-Fatt
authors.initials: KF
authors.lastname: Low
authors.forename: Abdah
authors.initials: A
authors.lastname: Karimah
authors.forename: Chan Yean
authors.initials: CY
authors.lastname: Yean
mesh_terms: D001483:Base Sequence; D015374:Biosensing Techniques; D002771:Cholera / Q000175:diagnosis* / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics / Q000302:isolation & purification*; D005243:Feces / Q000382:microbiology; D006801:Humans; D059346:Magnets; D009693:Nucleic Acid Hybridization; D014734:Vibrio cholerae / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D004269:DNA, Bacterial