Centre : Central Citrus Research Institute, Nagpur
Name of the Protocol
Detection of Phytophthora nicotianae in irrigation water by Ypt1 gene based nested PCR
Nature : Modification of existing protocol
Crop / Pathogen : Citrus / Phytophthora nicotianae
A set of universal primer pair Ypt1F/Ypt1R (outer set) and P. nicotianae specific primers Pn1/Pn2 (inner set) from regions of the RAS-related protein (Ypt1) were used. Amplifications were performed BIORAD T100TM Thermal Cycler PCR machine. PCR reactions were carried out in a 25µl reaction volume containing 2.5 μl of 10X PCR buffer (Fermentas Inc. Maryland, USA), 0.5 μl of 2.5 mM dNTPs, 2.5 μl of 25 mM MgCl2, 2.5 μl each of 10 µm primers (universal primers Ypt1F and Ypt1R) (Integrated DNA Technologies, USA), 0.2 μl of 20 mg/ml BSA (Fermentas Inc. Maryland, USA) and 1.25 U of Taq DNA polymerase (Invitrogen, Life Technologies Corporation, USA ), 1 μl template DNA (20 - 40 ng/µl). The thermal cycling conditions used was: initial denaturation at 94°C for 5 min, followed by 35 cycles of denaturation, annealing and elongation steps respectively at 94°C for 30 sec, 58°C for 30 sec and 72°C for 1 min and a final extension step of 72ºC for 10 min. The PCR conditions used for inner primer Pn1/Pn2 were similar as described for Ypt1F/Ypt1R, but with the exception of an annealing temperature of 66°C, 30 cycles and exclusion of BSA from the reaction mixture.