Mohammad Amin Almasi and Seyedmohammad Hosseyni Dehabadi
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.