A thermostabilized magnetogenosensing assay for DNA sequence-specific detection and quantification of Vibrio cholerae Kim-Fatt Low, Abdah Karimah, Chan Yean Yean n Department of Medical Microbiology and Parasitology, School of Medical Sciences, Universiti Sains Malaysia,16150 Kubang Kerian, Kelantan, Malaysia article info Article history: Received 29 November 2012 Received in revised form 4 March 2013 Accepted 5 March 2013 Available online 14 March 2013 Keywords: Vibrio cholerae Thermostabilized Magnetic bead Gold nanoparticle Electrochemical genosensor Asymmetric PCR amplicon 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 nanoparti- cles 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 1C). 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 3.9 nM, 5 pg/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 o10%, demonstrating its reliability and accuracy. & 2013 Elsevier B.V. All rights reserved. 1. Introduction Vibrio cholerae is classified into three broad serogroups: O1, O139, and non-O1/non-O139 (Baron et al., 2007). Both V. cholerae O1 and O139 are the etiological agents of epidemic and pandemic cholera owing to their toxigenicities. Cholera is an acute infectious disease characterized by rapid onset of severe secretory diarrhea with the production of “rice water” stools (Ghose, 2011). Without immediate rehydration treatment, death by fluid loss can occur within hours or days (Weil et al., 2012). V. cholerae non-O1/non- O139 typically do not produce cholera toxin and is nonepidemic, but can be pathogenic marked by incidents of extraintestinal infections, bacteremia (Petsaris et al., 2010), and sporadic out- breaks of acute gastroenteritis (Khuntia et al., 2008; Punpanich et al., 2011). The gold standard method for V. cholerae identification in clinical and environmental samples is by classical isolation proce- dures (Bopp et al., 1999; Choopun et al., 2002) consisting of cultural, biochemical, and immunological assays. However, this classical methodology is time-consuming (requiring several days to complete), difficult, and labor-intensive. Due to the virulence of V. cholerae, the development of a low-cost, rapid, sensitive, and specific analytical method is extremely important and highly desirable. To this end, an electrochemical genosensing (DNA-based electrochemical biosensing) assay would be an ideal approach to identify pathogenic V. cholerae. It is because this assay has inherent specificity and selectivity provided by nucleic acid sequence- specific hybridization as well as the advantages offered by electrochemical-based biosensing technology such as cost effec- tiveness, rapid and accurate analysis, simplicity, possibility of miniaturization, and the capability of point-of-care diagnosis. The use of electrochemical genosensing for sequence-specific detection of pathogenic microorganisms using different detection strategies has drastically increased in recent years (Campuzano et al., 2011; Geng et al., 2011; Siddiquee et al., 2010). The use of nanoparticles as labels in electrochemical genosensing systems is well established (Castañeda et al., 2007; Merkoçi, 2007). Among them, the application of gold nanoparticles (AuNPs) in biosensing assays has been of particular interest owing to its favorable characteristics such as facile chemical synthesis with size- controllability, good stability with a relatively defined nano-scaled Contents lists available at SciVerse ScienceDirect journal homepage: www.elsevier.com/locate/bios Biosensors and Bioelectronics 0956-5663/$ - see front matter & 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bios.2013.03.004 n Corresponding author. Tel.: þ609 7676258, þ609 7676275; fax: þ609 7641615. E-mail addresses: lowkimfatt@hotmail.my, lowkimfatt2089@yahoo.com (K.-F. Low), karimah@kb.usm.my (A. Karimah), yeancyn@yahoo.com (C.Y. Yean). Biosensors and Bioelectronics 47 (2013) 38–44