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Assessment of Performance Ability of Three Diagnostic Methods for Detection of Potato Leafroll Virus (PLRV) Using Different Visualizing Systems

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Abstract

To diminish the time required for some diagnostic assays including reverse transcription PCR (RT-PCR), reverse transcription loop-mediated isothermal amplification (RT-LAMP; due to mainly RNA extraction step) and also DAS-ELISA into a minimum level, an innovative immunocapture RT-LAMP (IC–RT-LAMP) and immunocapture reverse transcription (IC/RT-PCR) protocol on the basis of Potato Leafroll virus (PLRV) genome were used and optimized. In this regard, all six IC–RT-LAMP primers (i.e. F3, B3, FIP, BIP, LF and LB) together with IC/RT-PCR primers were designed on the basis of the highly conserved sequence (ORF3) of coat protein gene (GenBank accession number: U73777) of PLRV genome. Even though DAS-ELISA, IC/RT-PCR and IC–RT-LAMP assays could successfully detect positive infected plant samples, considering the time, safety, sensitivity, cost and simplicity, the last one was overall superior. Meanwhile, among five different visual dyes to accurately detect IC–RT-LAMP products, both hydroxynaphthol blue and GeneFinderTM could produce long stable colour change and brightness in a close tube-based approach to prevent cross-contamination risk, concluded eventually as the best ones. Altogether, as IC–RT-LAMP is sensitive, cost-effective, fairly user friendly and also can generate more accurate results than previous diagnostic procedures, we accordingly propose this colorimetric assay as a highly reliable alternative viral recognition system regarding PLRV recognition and probably other viral-based diseases.

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Abbreviations

CP:

Coat protein

DAS-ELISA:

Enzyme-linked immunosorbent assay

HNB:

Hydroxynaphthol blue

IC–RT-LAMP:

Immunocapture reverse transcription loop-mediated isothermal amplification

IC–RT-PCR:

Immunocapture reverse transcription polymerase chain reaction

LAMP:

Loop-mediated isothermal amplification

PLRV:

Potato Leafroll virus

RT-LAMP:

Reverse transcription loop-mediated isothermal amplification

RT-PCR:

Reverse transcription polymerase chain reaction

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Acknowledgments

The fund of performing the present investigation was provided by Payam Noor University of Ahvaz-Iran, Financial Division; accordingly, the authors would like to appreciate them for their financial support.

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Correspondence to Jaber Nasiri.

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The first three authors (i.e., Mohammad Amin Almasi & Aboubakr Moradi & Jaber Nasiri) contribute equally to this work.

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Almasi, M.A., Moradi, A., Nasiri, J. et al. Assessment of Performance Ability of Three Diagnostic Methods for Detection of Potato Leafroll Virus (PLRV) Using Different Visualizing Systems. Appl Biochem Biotechnol 168, 770–784 (2012). https://doi.org/10.1007/s12010-012-9818-1

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  • DOI: https://doi.org/10.1007/s12010-012-9818-1

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