Development of an electrochemical method for Ochratoxin A detection based on aptamer and loop-mediated isothermal amplification.

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.

[1]  P. Karanis,et al.  Loop-mediated isothermal amplification (LAMP) for malarial parasites of humans: would it come to clinical reality as a point-of-care test? , 2012, Acta tropica.

[2]  Yan Du,et al.  An integrated sensing system for detection of DNA using new parallel-motif DNA triplex system and graphene--mesoporous silica--gold nanoparticle hybrids. , 2011, Biomaterials.

[3]  Yun Xiang,et al.  Ultrasensitive aptamer-based protein detection via a dual amplified biocatalytic strategy. , 2010, Biosensors & bioelectronics.

[4]  A. Marx,et al.  Genotyping--from genomic DNA to genotype in a single tube. , 2005, Angewandte Chemie.

[5]  Zhengping Li,et al.  One-step ultrasensitive detection of microRNAs with loop-mediated isothermal amplification (LAMP). , 2011, Chemical communications.

[6]  Kevin W Plaxco,et al.  Rapid, sensitive, and quantitative detection of pathogenic DNA at the point of care through microfluidic electrochemical quantitative loop-mediated isothermal amplification. , 2012, Angewandte Chemie.

[7]  Eric S McLamore,et al.  Nanomaterial based self-referencing microbiosensors for cell and tissue physiology research. , 2013, Biosensors & bioelectronics.

[8]  Mohammadali Safavieh,et al.  Real-time electrochemical detection of pathogen DNA using electrostatic interaction of a redox probe. , 2013, The Analyst.

[9]  D.H. Kim,et al.  Rapid and sensitive detection of Streptococcus iniae by loop-mediated isothermal amplification (LAMP). , 2011, Journal of fish diseases.

[10]  Chun-yang Zhang,et al.  Sensitive detection of microRNAs with hairpin probe-based circular exponential amplification assay. , 2012, Analytical chemistry.

[11]  Ronghua Yang,et al.  Rolling circle amplification combined with gold nanoparticle aggregates for highly sensitive identification of single-nucleotide polymorphisms. , 2010, Analytical chemistry.

[12]  Tom Brown,et al.  Fluorescence based strategies for genetic analysis. , 2005, Chemical communications.

[13]  S. Ueda,et al.  The rapid detection of Salmonella from food samples by loop-mediated isothermal amplification (LAMP). , 2009, Biocontrol science.

[14]  B. Ye,et al.  Attomolar ultrasensitive microRNA detection by DNA-scaffolded silver-nanocluster probe based on isothermal amplification. , 2012, Analytical chemistry.

[15]  Peter J. Asiello,et al.  Miniaturized isothermal nucleic acid amplification, a review. , 2011, Lab on a chip.

[16]  Xi Chen,et al.  Adapting enzyme-free DNA circuits to the detection of loop-mediated isothermal amplification reactions. , 2012, Analytical chemistry.

[17]  Lishu Zheng,et al.  Detection of enterovirus 71 using reverse transcription loop-mediated isothermal amplification (RT-LAMP). , 2012, Journal of virological methods.

[18]  T. Notomi,et al.  Loop-mediated isothermal amplification of DNA. , 2000, Nucleic acids research.

[19]  H Otteneder,et al.  Occurrence of ochratoxin A (OTA) in wines: influence of the type of wine and its geographical origin , 2000, Food additives and contaminants.

[20]  Wenwan Zhong,et al.  Typing of multiple single-nucleotide polymorphisms by a microsphere-based rolling circle amplification assay. , 2007, Analytical chemistry.

[21]  P. Gill,et al.  Nucleic Acid Isothermal Amplification Technologies—A Review , 2008, Nucleosides, nucleotides & nucleic acids.

[22]  Yasuyoshi Mori,et al.  Loop-mediated isothermal amplification (LAMP) of gene sequences and simple visual detection of products , 2008, Nature Protocols.

[23]  Jian Xie,et al.  An aqueous media based approach for the preparation of a biosensor platform composed of graphene oxide and Pt-black. , 2012, Biosensors & bioelectronics.

[24]  Chen-Zhong Li,et al.  Impedance sensing of DNA binding drugs using gold substrates modified with gold nanoparticles. , 2005, Analytical chemistry.

[25]  Masato Saito,et al.  Semi-real time electrochemical monitoring for influenza virus RNA by reverse transcription loop-mediated isothermal amplification using a USB powered portable potentiostat. , 2011, The Analyst.

[26]  Chunhai Fan,et al.  Catalytic gold nanoparticles for nanoplasmonic detection of DNA hybridization. , 2011, Angewandte Chemie.

[27]  Hui Chen,et al.  Predicting viruses accurately by a multiplex microfluidic loop-mediated isothermal amplification chip. , 2011, Analytical chemistry.

[28]  Manmohan Parida,et al.  Loop mediated isothermal amplification (LAMP): a new generation of innovative gene amplification technique; perspectives in clinical diagnosis of infectious diseases , 2008, Reviews in medical virology.

[29]  K. Silamut,et al.  Loop-mediated isothermal PCR (LAMP) for the diagnosis of falciparum malaria. , 2007, The American journal of tropical medicine and hygiene.

[30]  T. Komuro,et al.  Rapid and simple sex determination method from dental pulp by loop-mediated isothermal amplification. , 2008, Forensic science international. Genetics.

[31]  J. Ndung'u,et al.  Loop-Mediated Isothermal Amplification (LAMP) Method for Rapid Detection of Trypanosoma brucei rhodesiense , 2008, PLoS neglected tropical diseases.