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Matrix approach to the simultaneous detection of multiple potato pathogens by real‐time PCR
Author(s) -
Nikitin M.M.,
Statsyuk N.V.,
Frantsuzov P.A.,
Dzhavakhiya V.G.,
Golikov A.G.
Publication year - 2018
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.13686
Subject(s) - ralstonia solanacearum , clavibacter michiganensis , biology , pectobacterium carotovorum , phytophthora infestans , potato virus y , pathogen , pectobacterium , microbiology and biotechnology , polymerase chain reaction , dna microarray , blackleg , blight , virology , virus , plant virus , gene , horticulture , genetics , gene expression , brassica
Aim Create a method for highly sensitive, selective, rapid and easy‐to‐use detection and identification of economically significant potato pathogens, including viruses, bacteria and oomycetes, be it single pathogen, or a range of various pathogens occurring simultaneously. Methods and Results Test‐systems for real‐time PCR , operating in the unified amplification regime, have been developed for Phytophthora infestans , Pectobacterium atrosepticum , Dickeya dianthicola , Dickeya solani , Ralstonia solanacearum , Pectobacterium carotovorum , Clavibacter michiganensis subsp. sepedonicus , potato viruses Y (ordinary and necrotic forms as well as indiscriminative test system, detecting all forms), A, X, S, M, potato leaf roll virus, potato mop top virus and potato spindle tuber viroid. The test‐systems (including polymerase and revertase) were immobilized and lyophilized in miniature microreactors (1·2 μ l) on silicon DNA / RNA microarrays (micromatrices) to be used with a mobile Aria DNA ® amplifier. Conclusions Preloaded 30‐reaction micromatrices having shelf life of 3 and 6 months (for RNA ‐ and DNA ‐based pathogens, respectively) at room temperature with no special conditions were successfully tested on both reference and field samples in comparison with traditional ELISA and microbiological methods, showing perfect performance and sensitivity (1 pg). Significance and Impact of the Study The accurate, rapid and user‐friendly diagnostic system in a micromatrix format may significantly contribute to pathogen screening and phytopathological studies.