Centre : Central Citrus Research Institute, Nagpur
Name of the Protocol
Development of conventional and Real-Time PCR assays for rapid and sensitive detection of P. nicotianae causing root rot and gummosis of citrus
Nature : Modification of existing protocol
Crop / Pathogen : Citrus / Phytophthora nicotianae
Conventional-PCR: A set of ITS region-based primer pair (PNIC1/PNIC2) specific to Phytophthora nicotianae was used. PCR amplifications were carried out on BIORAD T100TM Thermal Cycler in a 25µl volume consisting 1X PCR buffer, 2 mM MgCl2, 0.2 mM each dNTP, 0.5 µM of each primer (PNIC1/PNIC2), 1 U of Taq DNA polymerase (Bioline Inc, USA) and 1 µl of template DNA. The PCR conditions used were: initial denaturation at 94°C for 3 min, followed by 35 cycles of denaturation, annealing and elongation steps respectively at 94°C for 30 sec, 60°C for 30 sec and 72°C for 1 min and final extension step of 72ºC for 10 min. Real-Time-qPCR: ITS region primer pair (Ph.nicF/Ph.nicR) and probe (Pn.pro-FAM) specific to P. nicotianae were used. The Real-time PCR amplification reaction was performed using a ABI 7300 Real Time PCR system (Applied Biosystems, California) in a 25 µl reaction volume consisting of 200 nM (each) primer, 100 nM probe, 1X TaqMan® universal Master Mix (Applied Biosystems, CA) and 1 µl of template DNA. The standard amplification protocol was: The standard amplification protocol was 95°C for 15 min followed by 40 cycles at 94°C for 15 sec and 60°C for 1 min.