Protocol

Centre : Central Potato Research Institute(CPRI)
Name of the Protocol
Simultaneous detection of early and late blight of potato.
Nature : Novel
Crop / Pathogen : Potato/ Phytophthora infestans and Alternaria solani.

Inventors

1. Touseef Hussain
2. BP Singh
3. S K Kaushik
4. Mehi Lal
5. Anubha Gupta

Objectives

1. Simultaneous detection of early and late blight of potato in a single PCR.

Procedure / Summary

PCR reaction was performed in a total volume of 25 µl, contacting of 2.0µl 10X GreenTaq Buffer (with 1.5mM MgCl2), 2.0µl dNTPs (2.5mM), 2.0µl both pathogen specific primers 10µM(F+R), 0.2µl Taq DNA Polymerase Enzyme(5U) (Fermentas), 2.0 µl Template DNA, and 9.8µL Double distilled water. The PCR reaction was performed in thermal cycler (Eppendorff Mastercycler). The PCR conditions are as follows: denaturation at 94 °C for 2 min, followed by 30 cycles of 94 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min and a final extension at 72 °C for 10 min. Amplified PCR products (524 bp for Phytophthora infestans & 289 bp for Alternaria solani) were separated by electrophoresis on 2.0 % agarose gel and picture was captured under gel documentation system.

Applications

1. Simultaneous detection of early and late blight of potato in a single PCR.

Publications

1. Touseef Hussain, B. P. Singh, S.K Kaushik, Mehi Lal and Anubha Gupta (2014). PCR protocol for quick and combined detection of early and late blights of potato p169. National Seminar on emerging problems of potato (1-2 November 2014, CPRI Shimla).