ABSTRACT
Based on the sequence of the ochratoxin A polyketide synthase gene (otapksPV), a polymerase chain reaction (PCR) system for the specific detection of Penicillium verrucosum in wheat has been developed. In a further approach, a real-time PCR system has been applied to determine the growth kinetics of P. verrucosum in wheat at cell numbers above 10(3) colony-forming units (cfu) ml(-1). The data obtained by real-time PCR correlated well with the data obtained by the plate count technique. For this purpose, the DNA was isolated directly from contaminated wheat without any further enrichment step. In a reverse transcriptase real-time PCR, the expression of the otapksPV gene in wheat was detected 22 days after inoculation and storage at ambient temperature. Reasonable amounts of ochratoxin A, however, could not be detected before day 30. This early activation of ochratoxin A related genes was confirmed by microarray analysis.
Subject(s)
Food Microbiology , Ochratoxins/biosynthesis , Penicillium/metabolism , Triticum/microbiology , DNA, Fungal/genetics , Food Handling/methods , Gene Expression , Gene Expression Profiling/methods , Humans , Ochratoxins/analysis , Oligonucleotide Array Sequence Analysis , Penicillium/genetics , Penicillium/growth & development , Polymerase Chain Reaction/methods , TimeABSTRACT
The growth and ochratoxin A producing activity at the phenotypical and molecular level ofP. verrucosum in wheat stored at different relative moisture conditions have been followed. Growth has been measured by determination of colony forming units (cfu). The expression of the ochratoxin A polyketide synthase (otapksPV) has been followed over time by reverse transcriptase Real Time PCR, whereas the biosynthesis of ochratoxin A by the fungus has been followed by HPLC. The remoisted and inoculated wheat was kept in perforated plastic bags and stored in tower silos. The bags were adjusted to 24%, 19% and 14% (m/m) relative moisture and stored for 6 month (September 06 - February 06) at ambient temperatures. A high increase of the cfu number up to a level of about lx10(8) cfu/g could be observed in the 24% sample. Interestingly no growth could be detected in the 14% and 19% samples. This result was supported by the quantitative Real Time PCR data. In congruence with this growth behaviour ochratoxin A could be detected from the first to the last time point in the 24% sample. Interestingly the ochratoxin A amounts measured over time did not show increasing or constant values, but varied from higher to lower values. After storage of three month (November 2006) the ochratoxin A concentration in the wheat was highest. The expression of theotapksPV gene mirrors this behaviour. The expression of this gene also fluctuates around a certain value, showing high activity after three month of storage.
ABSTRACT
The application of headspace solid-phase microextraction for isolation and enrichment of solvent residues from oils and pharmaceuticals is discussed. The optimal parameters for isolation and preconcentration of common process solvents (hexane, benzene, toluene and selected chloroderivatives of hydrocarbons) were established. Four fiber types (100 microm polydimethylsiloxane (PDMS), 75 microm Carboxen-PDMS, 65 microm PDMS-divinylbenzene and 85 microm polyacrylate) were evaluated to choose the most efficient coating, able to absorb the greatest amount of analytes. GC-flame ionization detection (FID) and GC-electron-capture detection systems were used for quantitative and qualitative analysis, adequately to the appropriate group of the analytes. For all compounds the limit of detection (LOD), linearity, dynamic range, repeatability and intermediate precision were estimated.
