Protocol

Centre : Indian Agricultural Research Institute,New DelhI
Name of the Protocol
Detection of Ralstonia solanacearum from Asymptomatic Tomato Plants, Irrigation Water, and Soil Through Non-selective Enrichment Medium with hrp Gene-Based Bio-PCR
Nature : Novel
Crop / Pathogen : tomato/Ralstonia solanacearum

Inventors

1. Dinesh Singh
2. Shweta Sinha
3. D. K. Yadav
4. Garima Chaudhary

Objectives

1. Detection of R. solanacearum from Asymptomatic Plants, Irrigation Water and Soil
2. Detection of R. solanacearum from Irrigation Water and Soil

Procedure / Summary

The reaction was performed in a final volume of 20 ll amplification reaction mixture containing Buffer 2.0 ll (5X), Primer F (20 pmol/ll) 0.4 ll, Primer R (20 pmol/ll) 0.4 ll, MgCl2 (25 mM) 1.0 ll, dNTPs (10 mM) 0.4 ll, Taq polymerase 0.25 ll. Amplification conditions included, denaturation step at 95 C for 2 min followed by 35 cycles at 95 C for 30 s, 51 C for 30 s, and 72 C for 30 s, and then one cycle of 72 C for 10 min in Thermal cycler gradient PCR (BIO-RAD; model: C1000TM Thermal Cycler). Amplified PCR products were separated by electrophoresis on 1.2 % agarose gel (80 V) for 1 h and visualized UV light (300 nm) after staining with ethidium bromide under gel documentation system

Applications

1. The methods, are able to detect R. solanacearum from primary source inoculum like soil, water, and plants with enrichment steps definitely would be faster and have less PCR inhibitors particularly when bacterial DNA template is taken from soil suspension, bacterial ooze, and broth culture without DNA extraction.

Publications

1. Curr Microbiol (2014) 69:127–134 DOI 10.1007/s00284-014-0566-z