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Adapting enzyme-free DNA circuits to the detection of loop-mediated isothermal amplification reactions.

ABSTRACT: Loop-mediated isothermal amplification of DNA (LAMP) is a powerful isothermal nucleic acid amplification technique that can accumulate ~10(9) copies from less than 10 copies of input template within an hour or two. Unfortunately, while the amplification reactions are extremely powerful, the quantitative detection of LAMP products is still analytically difficult. In this article, to both improve the specificity of LAMP detection and to make direct readout of LAMP amplification simpler and much more reliable, we have developed a nonenzymatic nucleic acid circuit (catalyzed hairpin assembly, CHA) that can both amplify and integrate the specific sequence signals present in LAMP amplicons. Through a hairpin acceptor, one of the four loop products amplified from the LAMP is transduced to an active catalyst ssDNA which can in turn trigger a CHA reaction. After CHA detection, even less than 10 molecules/?L model templates (M13mp18) can produce significant signal, and both nonspecific template and parasitic amplicons cannot bring interference at all. More importantly, to further enhance the specificity, we have designed a dual-CHA circuit that only gave positive responses in presence of two LAMP loops. The AND-GATE detector will act as a simultaneous, specific readout of the LAMP product, rather than of competing and parasitic amplicons.

SUBMITTER: Li B

PROVIDER: S-EPMC3478682 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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Adapting enzyme-free DNA circuits to the detection of loop-mediated isothermal amplification reactions.

Li Bingling B Chen Xi X Ellington Andrew D AD

Analytical chemistry 20120914 19

Loop-mediated isothermal amplification of DNA (LAMP) is a powerful isothermal nucleic acid amplification technique that can accumulate ~10(9) copies from less than 10 copies of input template within an hour or two. Unfortunately, while the amplification reactions are extremely powerful, the quantitative detection of LAMP products is still analytically difficult. In this article, to both improve the specificity of LAMP detection and to make direct readout of LAMP amplification simpler and much mo ...[more]

PMID: 22947054

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Project description:Background:Enterocytozoon hepatopenaei (EHP) is a newly emerged microsporidian parasite that causes retarded shrimp growth in many countries. But there are no effective approaches to control this disease to date. The EHP could be an immune risk factor for increased dissemination of other diseases. Further, EHP infection involves the absence of obvious clinical signs and it is difficult to identify the pathogen through visual examination, increasing the risk of disease dissemination. It is urgent and necessary to develop a specific, rapid and sensitive EHP-infected shrimp diagnostic method to detect this parasite. In the present study, we developed and evaluated a rapid real-time loop-mediated isothermal amplification (real-time LAMP) for detection of EHP. Methods:A rapid and efficient real-time LAMP method for the detection of EHP has been developed. Newly emerged EHP pathogens in China were collected and used as the sample, and three sets of specificity and sensitivity primers were designed. Three other aquatic pathogens were used as templates to test the specificity of the real-time LAMP assay. Also, we compared the real-time LAMP with the conventional LAMP by the serial dilutions of EHP DNA and their amplification curves. Application of real-time LAMP was carried out with clinical samples. Results:Positive products were amplified only from EHP, but not from other tested species, EHP was detected from the clinical samples, suggesting a high specificity of this method. The final results of this assay were available within less than 45 min, and the initial amplification curve was observed at about 6 min. We found that the amplification with an exponential of sixfold dilutions of EHP DNA demonstrated a specific positive signal by the real-time LAMP, but not for the LAMP amplicons from the visual inspection. The real-time LAMP amplification curves demonstrated a higher slope than the conventional LAMP. Discussion:In this study, pathogen virulence impacts have been increased in aquaculture and continuous observation was predominantly focused on EHP. The present study confirmed that the real-time LAMP assay is a promising and convenient method for the rapid identification of EHP in less time and cost. Its application greatly aids in the detection, surveillance, and prevention of EHP.

Dataset Information

Adapting enzyme-free DNA circuits to the detection of loop-mediated isothermal amplification reactions.

Publications

Adapting enzyme-free DNA circuits to the detection of loop-mediated isothermal amplification reactions.

Similar Datasets

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