A thermostabilized magnetogenosensing assay for DNA sequence-specific detection and quantification of Vibrio cholerae

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• 作者：Kim-Fatt Low ^{lowkimfatt@hotmail.my lowkimfatt2089@yahoo.com} ; Abdah Karimah ^{karimah@kb.usm.my} ; Chan Yean Yean ; ^{yeancyn@yahoo.com}
• 关键词：Vibrio cholerae ; Thermostabilized ; Magnetic bead ; Gold nanoparticle ; Electrochemical genosensor ; Asymmetric PCR amplicon
• 刊名：Biosensors and Bioelectronics
• 出版年：2013
• 出版时间：15 September, 2013
• 年：2013
• 卷：47
• 期：Complete
• 页码：38-44
• 全文大小：863 K

文摘

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 ¡ãC). 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/¦Ìl, and 10³ 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.