Temperature inactivation of Feline calicivirus vaccine strain FCV F-9 in comparison with human noroviruses using an RNA exposure assay and reverse transcribed quantitative real-time polymerase chain reaction-A novel method for predicting virus infectivity.

A one-step reverse transcription quantitative real-time polymerase chain reaction (RT-QPCR) method in combination with RNase treatment and low copy number samples was developed in order to examine the effect of temperature on the ability of virus capsids to protect their RNA content. The method was applied to a non-cultivable virus (GII.4 norovirus) and Feline calicivirus vaccine strain F-9 (FCV) which is often used as a norovirus surrogate. Results demonstrated that FCV RNA is exposed maximally after 2min at 63.3 degrees C and this correlated with a greater than 4.5log reduction in infectivity as assessed by plaque assay. In contrast human GII.4 norovirus RNA present in diluted clinical specimens was not exposed maximally until 76.6 degrees C, at least 13.3 degrees C greater than that for FCV. These data suggest that norovirus possesses greater thermostability than this commonly used surrogate. Further, these studies indicate that current food processing regimes for pasteurisation are insufficient to achieve inactivation of GII.4 NoVs. The method provides a novel molecular method for predicting virus infectivity.

Correlation between DNA of trichothecene-producing Fusarium species and deoxynivalenol concentrations in wheat-samples.

AIMS: Correlations between DNA content of trichothecene-producing Fusarium spp. and concentration of the key mycotoxin deoxynivalenol (DON) in cereal samples. METHODS AND RESULTS: A LightCycler PCR-based assay was used to quantify the DNA from trichothecene-producing Fusarium spp. in 300 wheat samples. DNA concentrations ranged from not detectable to 16.3 mg kg-1 whereas DON concentrations (GC/MS data) varied from not detectable to 34.3 mg kg-1. Data analysis revealed a coefficient of correlation r=0.9557 between DON concentrations and DNA-amounts over all samples. An interval of confidence for P=95% was calculated based on samples with DON concentrations < or = 1.5 mg kg-1 (n=234). CONCLUSIONS: Quantification of 32 samples of Fusarium-contaminated wheat was performed within 45 min. Data analysis allowed estimation of DON contamination from quantitative PCR data in the wheat samples. SIGNIFICANCE AND IMPACT OF THE STUDY: The method described is useful for the screening of cereals in industrial quality control.

Real time detection of the tri5 gene in Fusarium species by lightcycler-PCR using SYBR Green I for continuous fluorescence monitoring.

LightCycler technology combines rapid in vitro amplification of DNA with real time detection and quantification of the amount of target molecules present in a sample. The system enables a 35-cycle PCR with 32 samples do be completed in 45 min, including quantification and identification of the product. It is therefore well suited for routine analysis of large numbers of samples in quality control and for defining HACCP concepts. Based on PCR primers specific to the tri5 gene, a quantitative group specific assay was established for Fusarium species producing trichothecenes. In the assay, SYBR Green I was used as fluorescent dye enabling real time detection of PCR products. Characterisation of the amplicons was achieved by melting point analysis (85 +/- 0.1 degrees C). Nonspecific products such as primer dimers could readily be distinguished from the product by their lower melting points. Composition of the amplification buffer was optimised and various hot start methods were tested in order to achieve the highest sensitivity of the assay. Uracil DNA glycosylase was added to prevent amplification of nonspecific products due to DNA carryover. The spectrum of species detected was generally in accordance with the results found in conventional PCR using the Tox5 primer pair. Reproducibility in six parallel experiments of the assay was determined to be 98% in the range between 0.05 and 6 ng of purified Fusarium graminearum DNA. The assay was used to analyse 30 wheat samples contaminated with toxigenic Fusarium spp. Contamination ranged from 0% to 78% as revealed by mycological analysis, and this is compared with results from the LightCycler. This is the first report on the use of the LightCycler system in combination with SYBR Green I for the quantification and identification of fungal DNA in pure cultures and sample material.

Enzymatic halogenation catalyzed via a catalytic triad and by oxidoreductases.

During the search for haloperoxidases in bacteria we detected a type of enzymes that catalyzed the peroxide-dependent halogenation of organic substrates. However, in contrast to already known haloperoxidases, these enzymes do not contain a prosthetic group or metal ions nor any other cofactor. Biochemical and molecular genetic studies revealed that they contain a catalytic triad consisting of a serine, a histidine, and an aspartate. The reaction they catalyze is actually the perhydrolysis of an acetic acid serine ester leading to the formation of peracetic acid. As a strong oxidizing agent the enzymatically formed peracetic acid can oxidize halide ions, resulting in the formation of hypohalous acid which then acts as the actual halogenating agent. Since hypohalous acid is also formed by the heme- and vanadium-containing haloperoxidases, enzymatic halogenation catalyzed by haloperoxidases and perhydrolases in general lacks substrate specificity and regioselectivity. However, detailed studies on the biosynthesis of several halometabolites led to the detection of a novel type of halogenases. These enzymes consist of a two-component system and require NADH and FAD for activity. Whereas the gene for one of the components is part of the biosynthetic cluster of the halometabolite, the second component is an enzyme which is also present in bacteria from which no halometabolites have ever been isolated, like Escherichia coli. In contrast to haloperoxidases and perhydrolases the newly detected NADH/FAD-dependent halogenases are substrate-specific and regioselective and might provide ideal tools for specific halogenation reactions.