Protocol

Centre : Indian Agricultural Research Institute,New DelhI
Name of the Protocol
Development and application of Loop-Mediated Isothermal Amplification assay for detection of Ralstonia solanacearum- wilt pathogen of tomato plant
Nature : Novel
Crop / Pathogen : Tomato/ Ralstonia solanacearum

Inventors

1. Dr. Dinesh Singh
2. Garima Chaudhary
3. Dhananjay Kumar Yadav

Objectives

1. Development of LAMP assay for detection of Ralstonia solanacearum infecting tomato plamts.
2. Evaluation of sensitivity and specificity of LAMP assay.

Procedure / Summary

12.5µl Isothermal Master Mix and consisted of 5 primers, at the final conc. 0.2 μmol•L-1 each of F3 and B3, 2 μmol•L-1 each of FIP and BIP, l mol•L-1 dNTPs, l mol•L-1 betaine, 6 mmol•L-1 MgSO4, 2.5 μL 10× Bst-DNA Polymerase Buffer, 8 U Bst DNA polymerase and 5 μL template. All reactions for evaluation of the test were performed in a ThermoCycler instrument using -ve control and +ve control. The reaction time was optimized for 15, 30, 60, 90 and 120 min at 65°C, while the reaction temperature was optimized by incubating 60, 61, 62, 63, 64, 65 and 66°C for 60 min. The final standardized LAMP reactions were run at 60ºC for 60min. The concentrations of betaine and Mg2+ ion were optimized using a series conc. from 1 mol•L-1 to 4 mol•L-1 and from 1 mmol•L-1 to 7 mmol•L-1. The reaction terminated at 80°C for 10 min using dry bath cycler. The LAMP products were run on 2% agarose gels and stained with ethridium bromide. The possibility of LAMP detection of plant pathogenic bacteria i.e R. solanacearum using HNB was examined by dissolving in distilled H20 at 1.5 mM to prepare stock solution.

Applications

1. Loop-mediated Isothermal Amplification is a simple, rapid, specific and cost-effective nucleic acid amplification method.
2. Amplification and detection of gene can be completed in a single step, by incubating the mixture of samples, primers, DNA polymerase with strand displacement activity and substrates at a constant temperature (about 65°C).
3.  It provides high amplification efficiency, with DNA being amplified 109-1010 times in 15-60 minutes. Because of its high specificity, the presence of amplified product can indicate the presence of target gene.
4. New amplification method (No need of thermal cycler) used for pathogen detection in humans, animals and plants.
5.  Economy in cost as it does not require special reagents or sophisticated equipments.
6.  There is no need for a step to denature double stranded into a single stranded form.
7.  The whole amplification reaction takes place continuously under isothermal conditions.