title: Development of an electrochemical method for Ochratoxin A detection based on aptamer and loop-mediated isothermal amplification.
abstract: Loop-mediated isothermal amplification (LAMP) is an outstanding DNA amplification procedure, in which the reaction can accumulate 10(9) copies from less than 10 copies of input template within an hour. While the amplification reaction is extremely powerful, the quantitative detection of LAMP products is still analytically difficult. Besides, the type of targets that LAMP can detect is also less, which to some extent limited the application of LAMP. In this study, we are reporting for the first time an efficient and accurate detection system which employs the integration of LAMP, aptamer and the electrochemical method for the sensitive detection of Ochratoxin A (OTA). Aptamers were designed as the forward outer primer to trigger the LAMP reaction, and then the LAMP amplification products were combined with a redox active molecule methylene blue (MB) and analyzed by an electrode using differential pulse voltammograms (DPV). As the reaction progresses, the MB intercalated into double-stranded regions of LAMP amplicons reduces the free MB concentration. Hence, the peak current of reaction mixture decreased with the amplification because of the slow diffusion of MB-amplified DNA complex to the electrode surface. The peak height of the current was related to the input amount of the aptamers, providing a ready means to detection the concentration of OTA. With such design, the proposed assay showed a good linear relationship within the range of 0.001-50 nM with a detection limit of 0.3 pM (defined as S/N = 3) for OTA.
authors.affiliation: Key Laboratory of Ministry of Education on Luminescence and Real-Time Analysis, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China.
authors.forename: Shunbi
authors.initials: S
authors.lastname: Xie
authors.affiliation: Key Laboratory of Ministry of Education on Luminescence and Real-Time Analysis, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China. Electronic address: yqchai@swu.edu.cn.
authors.forename: Yaqin
authors.initials: Y
authors.lastname: Chai
authors.affiliation: Key Laboratory of Ministry of Education on Luminescence and Real-Time Analysis, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China.
authors.forename: Yali
authors.initials: Y
authors.lastname: Yuan
authors.affiliation: Key Laboratory of Ministry of Education on Luminescence and Real-Time Analysis, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China.
authors.forename: Lijuan
authors.initials: L
authors.lastname: Bai
authors.affiliation: Key Laboratory of Ministry of Education on Luminescence and Real-Time Analysis, School of Chemistry and Chemical Engineering, Southwest University, Chongqing 400715, PR China. Electronic address: yuanruo@swu.edu.cn.
authors.forename: Ruo
authors.initials: R
authors.lastname: Yuan
mesh_terms: D052157:Aptamers, Nucleotide / Q000737:chemistry* / Q000235:genetics*; D015374:Biosensing Techniques / Q000295:instrumentation*; D003217:Conductometry / Q000295:instrumentation*; D004867:Equipment Design; D019544:Equipment Failure Analysis; D021141:Nucleic Acid Amplification Techniques / Q000295:instrumentation*; D009793:Ochratoxins / Q000032:analysis* / Q000737:chemistry; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity; D013696:Temperature
chemical_list: D052157:Aptamers, Nucleotide; D009793:Ochratoxins; C025589:ochratoxin A
keywords: Aptamer; Electrochemical; Loop-mediated isothermal amplification; Ochratoxin A