Molecular typing of Aspergillus ochraceus and construction of species specific SCAR-primers based on AFLP.
70 strains of Aspergillus ochraceus mainly isolated from Brazilian coffee related sources were investigated for genetic relatedness using automated laser fluorescence analysis of AFLP fragments. Cluster analysis of fingerprints revealed a very close relationship among most of the strains. Based on these results, a sub-set of characteristic A. ochraceus strains was chosen for the detection of marker sequences. These sequences were obtained from silver stained AFLPs separated on polyacrylamide gels. A number of bands characteristic for A. ochraceus were detected and cut out from the gels. DNA was reamplified, cloned and fragments were sequenced. Based on these sequences a set of SCAR PCR-primers was constructed. PCRs were optimised for specificity and subsequently tested against a panel of Aspergillus species. Using this approach a PCR specific for Aspergillus ochraceus was developed.
TU München, Lehrstuhl für Technische Mikrobiologie, Freising, Germany.
Holger
H
Schmidt
Matthias
M
Ehrmann
Rudi F
RF
Vogel
Marta H
MH
Taniwaki
Ludwig
L
Niessen
D001235:Aspergillus ochraceus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification; D015373:Bacterial Typing Techniques / Q000379:methods*; D003001:Cloning, Molecular; D016172:DNA Fingerprinting / Q000379:methods*; D017931:DNA Primers; D005456:Fluorescent Dyes / Q000737:chemistry; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods*; D012150:Polymorphism, Restriction Fragment Length; D017422:Sequence Analysis, DNA; D016622:Silver Staining / Q000379:methods; D013045:Species Specificity
D017931:DNA Primers; D005456:Fluorescent Dyes