Colorimetric Immunocapture Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Rapid Detection of the Potato virus YMohammad Amin Almasi1 and Seyedmohammad Hosseyni Dehabadi2*
- *Corresponding Author:
- Seyedmohammad Hosseyni Dehabadi
Young Researchers and Elite Club, Meybod Branch
Islamic Azad University, Meybod, Iran
E-mail: [email protected]
Received date: June 24, 2013; Accepted date: July 11, 2013; Published date: July 15, 2013
Citation: Almasi MA, Dehabadi SH (2013) Colorimetric Immunocapture Reverse Transcription Loop-Mediated Isothermal Amplification Assay for Rapid Detection of the Potato virus Y. J Plant Pathol Microb 4:188 doi:10.4172/2157-7471.1000188
Copyright: © 2013 Almasi MA, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Loop-Mediated Isothermal Amplification (LAMP) assay is a novel technique for amplifying DNA under constant temperature, with high specificity, sensitivity, rapidity and efficiency. To diminish the time required for some diagnostic assays including Reverse Transcription PCR (RT-PCR), Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) and also DAS-ELISA into a minimum level, an innovative colorimetric IC-RT-LAMP and IC-RT-PCR protocol on the basis of Potato Virus Y (PVY) genome were used and optimized. Firstly, DAS-ELISA assay was performed to detect of the virus in a collection containing 95 suspicious samples. Lastly, five samples were detected as the positive samples. Then, the positive samples were verified by molecular methods. In this regard, all four RT-LAMP primers (i.e. F3, B3, FIP and BIP) together with RT-PCR primers (F and B) were selected on the basis of coat protein gene (CP) of PVY genome. Even though DAS-ELISA, RT-PCR and RT-LAMP assays could successfully detect positive infected plant samples, considering the time, safety, sensitivity, cost and simplicity, the last one was overall superior. Furthermore, the results demonstrated that the RT-LAMP assay was 100 times sensitive and 3 time faster compared to RT-PCR. LAMP assay was accomplished in the water bath either frees from any thermal cycler machine or sophisticated laboratories facility. Meanwhile, among six different visual dyes to accurately detect IC–RT-LAMP products, Hydroxynaphthol blue, GeneFinderTM and SYBR Green I 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. We accordingly propose this colorimetric assay as a highly reliable alternative viral recognition system regarding PVY recognition and probably other viral-based diseases.