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

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.