Development of a Loop-mediated Isothermal Amplification (Lamp) Assay for the Detection of Tomato Leaf Curl Virus in Potato (Solanum Tuberosum)

Abstract

Potato is a starchy, tuberous crop has emerged as the fourth important food crop in India. It is the most cultivated vegetables crops of India. Potato is a vulnerable host for abundant viral infectious agents in which Tomato Leaf curl New Delhi Virus (ToLCNDV) is the most important and its control measures are time consuming. Therefore, LAMP assay can be a potent diagnostic tool which offers rapid and easy detection. In the present study, 20 symptomatic and asymptomatic samples with Potato leaf curl were collected from Mansurpur, Khatauli, Sakoti, Daurala, Macchara and Pabli Khas villages nearby Meerut. Moecular diagnostics was performed using total genomic DNA extracted by CTAB method and PCR was performed using a set of specific primer for Coat Protein gene (TLCAV1F/AV1R) and expected size of ~ 770 bp was obtained. Amplification was obtained in 12 potato samples out of 20. The Positive samples were stored in -20 oC and further used for the development of LAMP assay. In order to perform LAMP assay, six ToLCNDV specific primers and cox (universal plant) gene specific primers were designed through LAMP Designer 1.13 software and synthesized. The Primers synthesized were tested using conventional PCR and through Real Time PCR with outer primers (F3 and B3). In conventional PCR the outer primers pairs from ToLCV and Cox gene produced promising results by amplifying DNA of ~230 bp as expected from the target. In Real Time PCR quantification was recorded with a PCR base line subtracted curve fit (RFU) against cycles with a base line threshold value of 17186.5. The melt curve of real time PCR with ToLCV infected samples had a single peak at 80-81 oC and a single peak was also obtained with cox primers used as control at 79 oC. The Ct for ToLCV value ranged 8.96 to11.45 and for cox 16.03 to 19.36. LAMP assay was performed with ToLCV specific primers, Bsm DNA polymerase, dNTPs and Betaine and produced multiple bands [ladder like Ppattern] of different sizes on agarose gel electrophoresis. The amplicons were also detected visually by adding ethidium bromide and HNB dyes which showed color changes in the tubes. To improve yield and specificity of LAMP assay different enhancing agents Betaine & DMSO were used. The result indicated that the combination of 5M betaine and 5% DMSO showed same yield as in 5M betaine. The sensitivity analysis of LAMP and conventional PCR showed that LAMP assay was more sensitive than conventional PCR. From this study we can be concluded that LAMP assay is a novel technique for rapid and easy detection as well as more sensitive than conventional PCR Furthermore, LAMP does not require expensive equipments (e.g., real-time PCR machine or a thermal cycler), and it may be performed successfully using a waterbath.