Protocol

Centre : Central Citrus Research Institute, Nagpur
Name of the Protocol
Multiplex PCR asaay for simultaneous detection of two Phytophthora pathogens, P. nicotianae and P. palmivora
Nature : Modification of existing protocol
Crop / Pathogen : Citrus / Phytophthora nicotianae, Phytophthora palmivora

Inventors

1. Das, A.K.
2. Nerkar, S.G.
3. Kadam, R.
4. Kumar, A.

Objectives

1. To develop multiplex PCR assay for simultaneous detection of Phytophthora nicotianae and P. palmivora

Procedure / Summary

Primers were designed specific for P. nicotianae (Pn1/Pn2) and P. palmivora (Pal1s ⁄ Pal2a ) based on Ypt1 gene and ITS region, respectively. Amplifications were performed BIORAD T100TM Thermal Cycler PCR machine. PCR reactions were carried out in a 25µl volume consisting of 1X PCR buffer (Fermentas Inc. Maryland, USA), 0.2 mM of each dNTPs (Fermentas Inc. Maryland, USA), 2 mM MgCl2 (Fermentas Inc. Maryland, USA), 0.5 µM of each (Pal1s/Pal2a) primers and 1 µM of each (Pn1/Pn2) primers (Integrated DNA Technologies, Coralville, USA), 1 U of Taq DNA polymerase (Invitrogen, Life Technologies Corporation, USA), 1 µl of template DNA and sterile distilled water was added to make up the final volume. The thermal cycling conditions used were: initial denaturation at 94°C for 5 min, followed by 30 cycles of denaturation, annealing and elongation steps respectively at 94°C for 30 sec , 65ºC for 30 sec and 72ºC for 1 min and a final extension step of 72ºC for 10 min.

Applications

1. The developed multiplex PCR assay substantiated to be sensitive and robust technique to detect P. nicotianae and P. palmivora simultaneously in case of mixed infection occurring in citrus nursery and field orchards.