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A rapid real‐time PCR/DNA resolution melting method to identify Prototheca species
Author(s) -
Ricchi M.,
Cammi G.,
Garbarino C.A.,
Buzzini P.,
Belletti G.L.,
Arrigoni N.
Publication year - 2011
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/j.1365-2672.2010.04861.x
Subject(s) - biology , genotype , high resolution melt , dna extraction , real time polymerase chain reaction , polymerase chain reaction , microbiology and biotechnology , genetics , gene
Aim: The study describes the development of a simple and rapid tool to identify yeast‐like microalgae belonging to the genus Prototheca. Methods and Results: The method, based on two‐step Real Time PCR reaction followed by DNA Resolution Melting Analysis (qPCR/RMA), has been developed using reference strains belonging to both pathogenic ( P. zopfii genotype 2, P. wickerhamii and P. blaschkeae ) and non ‐ pathogenic species ( P. zopfii genotype 1, P. stagnora and P. ulmea ). In order to validate the method, seventy recently isolated Prototheca strains were thus tested in parallel with both the first qPCR/RMA and the conventional genotype‐specific PCR assay: they were classified as P. zopfii genotype 1, P. zopfii genotype 2 and P. blaschkeae , with a perfect accordance between the two above methodologies. Furthermore, we used the second qPCR/RMA to identify the other species ( P. stagnora , P. ulmea and P. wickerhamii ), which cannot be discriminated by conventional PCR assay. Conclusions: The assay two‐step Real Time PCR is accurate, robust, cost‐effective and faster than auxonographical, biochemical or conventional molecular biology methods. Significance and Impact of the Study: the rapid and high throughout two‐step qPCR/RMA tool can be usefully used for the identification of clinical and environmental Prototheca species into the framework of the diagnosis of animal (e.g. bovine mastitis) or human protothecosis.