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Loop‐mediated isothermal amplification for detection of the tomato and potato late blight pathogen, P hytophthora infestans
Author(s) -
Hansen Z.R.,
Knaus B.J.,
Tabima J.F.,
Press C.M.,
Judelson H.S.,
Grünwald N.J.,
Smart C.D.
Publication year - 2016
Publication title -
journal of applied microbiology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.889
H-Index - 156
eISSN - 1365-2672
pISSN - 1364-5072
DOI - 10.1111/jam.13079
Subject(s) - blight , loop mediated isothermal amplification , phytophthora infestans , pathogen , biology , microbiology and biotechnology , horticulture , genetics , dna
Aims To design and validate a colorimetric loop‐mediated isothermal amplification assay for rapid detection of Phytophthora infestans DNA. Methods and Results Two sets of loop‐mediated isothermal amplification (LAMP) primers were designed and evaluated for their sensitivity and specificity for P. infestans . ITSII primers targeted a portion of the internal transcribed spacer region of ribosomal DNA. These primers had a limit of detection of 2 pg P. infestans DNA and cross‐reacted with the closely related species Phytophthora nicotianae . Rgn86_2 primers, designed to improve assay specificity, targeted a portion of a conserved hypothetical protein. These primers had a limit of detection of 200 pg P. infestans DNA and did not cross‐react with P. nicotianae . The specificity of the Rgn86_2 assay was tested further using the closely related species P. andina , P. ipomoeae , P. mirabilis and P. phaseoli . Cross‐reactions occurred with P. andina and P. mirabilis , but neither species occurs on tomato or potato. Both primer sets were able to detect P. infestans DNA extracted from tomato late blight leaf lesions. Conclusions Two colorimetric LAMP assays detected P. infestans DNA from pure cultures as well as infected leaf tissue. The ITSII primers had higher sensitivity, and the Rgn86_2 primers had higher specificity. Significance and Impact of the Study This is the first report of a LAMP assay for the detection of P. infestans , the causal organism of potato and tomato late blight. These assays have potential for immediate utility in plant disease research and diagnostic laboratories.