Development of a Real Time PCR system for detection of Penicillium nordicum and for monitoring ochratoxin A production in foods by targeting the ochratoxin polyketide synthase gene.
A 750 bp DNA fragment from the genome of Penicillium nordicum was isolated using degenerated primers for polyketide synthase genes. All analyzed P. nordicum strains possessed the fragment, whereas the closely related ochratoxinogenic P. verrucosum strains did not. The nucleotide sequence of the fragment has been determined. It shows homology to the pksL2 polyketide synthase gene from A. parasiticus. The deduced amino acid sequence of this fragment shows high homology to several other fungal polyketide synthases. An expression analysis of this gene by Reverse Transcription Real Time PCR demonstrates that this putative polyketide synthase gene (otapksPN) is highly induced under ochratoxin A producing conditions, but only to low levels under non-producing conditions. A Real Time PCR system based on the otapksPN sequence has been used to monitor growth and ochratoxin A production of P. nordicum in wheat. A strong correlation between the copy numbers of the otapksPN gene and the colony forming units (cfu) has been observed. In addition there was a strong congruence between otapksPN gene expression and ochratoxin A production in wheat. According to these results the fragment is obviously part of a polyketide synthase (otapksPN) which seems to be involved in the production of ochratoxin A.
Geisen
Rolf
R
Federal Research Centre for Nutrition and Food, Institute of Hygiene and Toxicology, Karlsruhe, Germany. rolf.geisen@bfe.uni-karlsruhe.de
Mayer
Zsuzsanna
Z
Karolewiez
Anja
A
Färber
Paul
P
D015169:Colony Count, Microbial; D009097:Multienzyme Complexes / Q000235:genetics / Q000378:metabolism; D009793:Ochratoxins / Q000096:biosynthesis* / Q000378:metabolism; D010407:Penicillium / Q000145:classification / Q000201:enzymology / Q000235:genetics / Q000302:isolation & purification*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D014908:Triticum / Q000382:microbiology*
D009097:Multienzyme Complexes; D009793:Ochratoxins; C025589:ochratoxin A