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a quantitative real time pcr based method for the detection of phytophthora infestans causing late blight of potato, in infested soil论文.pdf

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Saudi Journal of Biological Sciences (2014) 21, 380–386 King Saud University Saudi Journal of Biological Sciences .sa ORIGINAL ARTICLE A quantitative Real Time PCR based method for the detection of Phytophthora infestans causing Late blight of potato, in infested soil a, ,1 b c Touseef Hussain * , Bir Pal Singh , Firoz Anwar a Plant Pathology Lab., CPRI Campus, Modipuram 250110, Meerut City, U.P., India b Central Potato Research Institute, Shimla 171001, H.P., India c Siddhartha Institute of Pharmacy, Uttarakhand Technical University, Dehradun 248001, U.K., India Received 19 March 2013; revised 21 September 2013; accepted 29 September 2013 Available online 9 October 2013 KEYWORDS Abstract A fast and simple polymerase chain reaction method has been developed for detection of Detection; Phytophthora infestans oospores, the causal agent of Late blight of Potato in soil. The method ITS; involves the disruption of oospores by grinding dry soil, using abrasive properties, in the presence P. infestans; of glass powder and skimmed milk powder within a short time. The latter prevents loss of DNA by Oospores; adsorption to soil particles or by degradation and reduces the co-extraction of PCR inhibitors with PCR the DNA. After phenol/chloroform extraction; the DNA is suitable for direct PCR ampliﬁcation without a precipitation step. This ampliﬁcation leads