Open AccessSpecific Polymerase Chain Reaction Identification of Venturia nashicola Using Internally Transcribed Spacer Region in the Ribosomal DNA
Author(s) -
Bruno Le Cam,
Martine Devaux,
Luciana Parisi
Publication year - 2001
Publication title -
phytopathology
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.264
H-Index - 131
eISSN - 1943-7684
pISSN - 0031-949X
DOI - 10.1094/phyto.2001.91.9.900
Subject(s) - biology , primer (cosmetics) , polymerase chain reaction , restriction enzyme , ribosomal dna , genetics , genomic dna , internal transcribed spacer , microbiology and biotechnology , spacer dna , in silico pcr , oligonucleotide , restriction fragment length polymorphism , ribosomal rna , dna , nucleic acid sequence , multiplex polymerase chain reaction , gene , phylogenetics , chemistry , organic chemistry
A technique based on the polymerase chain reaction (PCR) was developed for the identification of Venturia nashicola using nucleotide sequence information of the ribosomal DNA region. The complete internal transcribed spacer (ITS) region of V. nashicola strains and phylo-genetically related species was amplified with the two universal ITS1 and ITS4 primers, sequenced, and digested with five restriction enzymes. The alignment of nucleotide sequences and analyses of digestion patterns indicated constant polymorphisms between V. nashicola and related species at nucleotides 126 and 127, which overlapped a TaqI restriction site. An oligonucleotide primer named A126 was designed for identifying this variable region. A primer set (A126 and ITS4) that allowed the amplification of a 391-bp DNA fragment within the ITS region by PCR was specific to V. nashicola when it was checked against fungal genomic DNAs of related fungi. This primer set was a good candidate for a species-specific reagent in a procedure for identification of V. nashicola by PCR.