Журнал патологии растений и микробиологии

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Development of Colorimetric Loop-Mediated Isothermal Amplification Assay for Rapid Detection of the Tomato Yellow Leaf Curl Virus

Mohammad Amin Almasi, Mehdi Aghapour Ojaghkandi, Ahmad Hemmatabadi, Fatah Hamidi and Saeedeh Aghaei

Loop-mediated isothermal amplification (LAMP) assay is a novel technique for amplifying DNA under constant temperature, with high specificity, sensitivity, rapidity and efficiency. We applied a rapid detection protocol, for the first time, which utilizes colorimetric loop-mediated isothermal amplification for detection of Tomato Yellow Leaf Curl virus. In this regard, all four LAMP primers (i.e. F3, B3, FIP and BIP,) together with PCR primers (F and R) were designed on the basis of the SF gene of the DNA sequences of TYLCV. Even though DAS-ELISA, PCR and LAMP assays could successfully detect positive infected samples, considering the time, safety, sensitivity, cost and simplicity, the last one was overall superior. Meanwhile, among five different visual dyes to accurately detect LAMP products, both hydroxynaphthol blue and GeneFinderTM could produce long stable color change and brightness in a close tube-based approach to prevent cross-contamination risk, concluded eventually as the best ones. All the results, overall, indicated that the LAMP offers an interesting novel and convenient assay format for the rapid,
sensitive, cost-effective, and fairly user friendly diagnostic tool of recognition of Tomato Yellow Leaf Curl virus and therefore presents an alternative to PCR-based assays.