Real-time PCR for Quantification of Toxigenic Fusarium Species in Barley and Malt

Abstract

A real-time PCR technique was applied for the quantification of trichothecene-producing Fusarium species (TMTRI assay) as well as the highly toxigenic Fusarium graminearum (TMFg12 assay) present in barley grain and malt. PCR results were compared to the amounts of trichothecenes detected in the samples to find out if the PCR assays can be used for trichothecene screening instead of expensive and laborious chemical analyses. DNA was extracted from ground kernels using a commercial DNA extraction kit and analysed in a LightCyclerÒ system using specific primers and fluorogenic TaqMan probes. Both naturally and artificially contaminated grains were analysed. The TMTRI assay and the TMFg12 assay enabled the quantification of trichothecene-producing Fusarium DNA and F. graminearum DNA present in barley grain and malt samples, respectively. Both TaqMan assays were considered to be sensitive and reproducible. Linearity of the assays was 4-5 log units when pure Fusarium DNAs were tested. The amount of Fusarium DNA analysed with the TMTRI-trichothecene assay could be used for estimation of the deoxynivalenol (DON) content in barley grain. Furthermore, the TMFg12 assay for F. graminearum gave a good estimation of the DON content in north American barley and malt samples, whilst the correlation was poor among Finnish samples. DON content and the level of F. graminearum DNA were found to be naturally low in Finnish barleys.

PCR detection of Fusarium fungi with similar profiles of the produced mycotoxins

“…Sarlin et al (2006) on Finnish barley and malt grain samples which contained relatively low DON levels. Low DON production in our samples can be explained due to strong dependence on the toxin production by such factors, as grain microflora, temperature, humidity, etc.…”

PCR detection and identification of Alternaria species-groups in processed foods based on the genetic marker Alt a 1

“…However, some authors have reported a positive correlation between the PCR detection of DNA from Fusarium culmorum and the presence of nivalenol in cereal samples (Pasquali et al, 2010). Also, Sarlin et al (2006) have reported a correlation between the F. graminearum DNA level and the deoxynivalenol content in north American barley and malt samples. A positive PCR result for mycotoxigenic Alternaria speciesgroups can therefore be taken as an indication of the use of low quality raw materials, and also the potential presence of Alternaria mycotoxins in the final products that should be analyzed further.…”

PCR-based assay for the detection of Alternaria species and correlation with HPLC determination of altenuene, alternariol and alternariol monomethyl ether production in tomato products

“…A positive correlation between the PCR detection of fungal DNA and mycotoxins has been reported for Fusarium culmorum and the presence of nivalenol in cereal samples (Pasquali et al, 2010). Also, Sarlin et al (2006) reported a correlation between Fusarium graminearum DNA concentration and the deoxynivalenol content in North American barley and malt samples.…”