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Comparison and Evaluation of Three Diagnostic Methods for Detection of Beet Curly Top Virus in Sugar Beet Using Different Visualizing Systems

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Abstract

To diminish the time required for some diagnostic assays including polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP; due to mainly DNA extraction step) and also triple antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) into a minimum level, an innovative immunocapture LAMP (IC-LAMP) and immunocapture PCR (IC-PCR) protocol on the basis of beet curly top virus (BCTV) genome was used and optimized. TAS-ELISA was employed first to validate the existence of the virus. All six IC-LAMP primers (i.e. forward outer primer (F3), backward outer primer (B3), forward inner primer (FIP), backward inner primer (BIP), loop forward (LF) and loop backward (LB)) together with IC-PCR primers were designed on the basis of the replication-associated protein (rep) gene (GenBank accession AF379637.1) of BCTV genome. Also, a novel colorimetric IC-LAMP assay for rapid and easy detection of BCTV was developed here, its potential compared with TAS-ELISA and IC-PCR assays. The method, on the whole, had the following advantages over the two mentioned procedures: (i) fascinatingly, no need of DNA extraction; (ii) no requirement of expensive and sophisticated tools for amplification and detection; (iii) no post-amplification treatment of the amplicons and (iv) a flexible and easy detection approach, which is visually detected by naked eyes using diverse visual dyes.

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Authors and Affiliations

  1. Department of Biotechnology, Research Institute of Physiology and Biotechnology, University of Zanjan, PO Box 45195-313, Zanjan, Iran

    Mohammad Amin Almasi

  2. Young Researchers and Elite Club, Meybod Branch, Islamic Azad University, Meybod, Iran

    Seyed Mohammad Hosseyni-Dehabadi

  3. Young Researchers and Elite Club, North Tehran Branch, Islamic Azad University, Tehran, Iran

    Mehdi Aghapour-ojaghkandi

Authors
  1. Mohammad Amin Almasi
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  2. Seyed Mohammad Hosseyni-Dehabadi
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  3. Mehdi Aghapour-ojaghkandi
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Correspondence to Mohammad Amin Almasi.

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Almasi, M.A., Hosseyni-Dehabadi, S.M. & Aghapour-ojaghkandi, M. Comparison and Evaluation of Three Diagnostic Methods for Detection of Beet Curly Top Virus in Sugar Beet Using Different Visualizing Systems. Appl Biochem Biotechnol 173, 1836–1848 (2014). https://doi.org/10.1007/s12010-014-0970-7

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  • DOI: https://doi.org/10.1007/s12010-014-0970-7

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