[Direct detection of T-2- and HT-2-mycotoxins producers of fungi the genus Fusarium in food grain by PCR (report 2)].

The improvement of the Fusarium DNA extraction method has been undertaken in order to reduce the error of PCR analysis for detection of toxigenic Fusarium species, including those contained in the grain in the uncultureted state, directly in the grain. The efficiency of Fusarium DNA extraction methods (nucleotides sorption and CTAB method) has been compared. The efficiency of CTAB method combined with 10-fold weight increase of milled grain sample has been demonstrated. This approach revealed a greater number of Fusarium species, than PCR analysis of combined Fusarium mycelium from the same samples. The uncultureted F. langsethiae was detected in the DNA extract from a sample of barley, which was not identified in the combined sample of the mycelium. This sample of the grain has the highest levels of T-2/NT-2-toxins--0,075/0,345 mg/kg (determined by HPLC) among positive samples. F. sporotrichioides--a potential producer of T-2- and HT-2-toxins has been revealed by PCR method in other grain samples both containing and not containing these toxins. The biosynthesis of T-2- and HT-2-toxins on the PSA-medium in vitro has been studied for 10 single-spores F. sporotrichioides isolates, allocated from grain. Synthesized T-2-toxin content (measured by ELISA) ranged from 0.4 to 184.5 mg per l of medium. Three strains showed very high levels from 117.2 to 184.4 mg/l, two of which have been isolated from barley which don't contain these toxins. The absence of the toxin in grain samples does not guarantee the absence of high-level producers of mycotoxins. The direct detection of Fusarium spp. in grain by PCR analysis with extraction of fungal DNA by CTAB method along with increased sample weight has been shown to make possible the detection of a more number of species of Fusarium (including uncultureol strains) compared with mycological method with PCR analysis of the combined sample of the mycelium.

Fusariotoxins in Russian Federation 2005-2010 grain harvests.

Monitoring results of food grain contamination with fusariotoxins-deoxynivalenol (DON), zearalenone (ZEN), fumonisins (FB1&FB2), T-2 and HT-2 toxins-are presented. Harvests of 2005-2010 in different regions of Russia were investigated. The occurrence of DON in wheat was 8%, barley 9%, oats 4%, rye 2% and maize 2%. The highest frequency of ZEN contamination was found in oats, the lowest in wheat. Calculated average daily intake of DON varied from 0.066 to 0.096 µg/kg body weight, the highest being found in the Southern region, but substantially lower than the provisional maximum tolerable daily intake. The results of enzyme-linked immunosorbent assay and high-performance liquid chromatography-mass spectrometry analysis demonstrated the presence of T-2 toxin in 14% and HT-2 toxin in 17% of all samples. The maximum level of T-2 toxin was exceeded only in one sample of barley. Relatively high frequency and levels of FB1&FB2 contamination were found in maize.

[Application of immunochemical and chromatographic techniques for monitoring of trichothecene mycotoxins type A in grains].

A sensitive HPLC-MS technique after immunoaffinity clean-up has been developed for the simultaneous determination of T-2 and HT-2 toxins of in the food grains. The limit of detection of method was 0,002 mg/kg for T-2 toxin and 0,005 mg/kg for HT-2 toxin. Comparison of existing immunochemical and chromatographic techniques of trichothecenes type A determination in grains was made. New combined scheme of monitoring of grain contamination by trichothecenes type A was proposed. It was based on screening by ELISAs followed by confirmation of positive samples by developed HPLC-MS technique with preliminary immune affinity purification of extracts. Proposed modification of immunochemical and chromatographic techniques allowed to lower the influence of grain matrix on the analytical results and to reduce of the probability obtaining of false-positive determination. Developed approach was used for determination of trichothecenes type A in natural contaminated grain samples.

[Fusariotoxins in the food production in the Russian Federation: situation 2006-2008 years].

The results of monitoring of the fusarium mycotoxins (deoxynivalenole (DON), zearalenone (ZL), T-2 and HT-2 toxins, fumonisins B1 and B2) contents in the food wheat, barley, rye, oats and corn (2006-2008 crops) in different regions of Russia were presented. DON occurrence in the food wheat, rye and barley were 9, 4 and 0,8% accordingly. DON wasn't detected in the food oats and maize in these years. ZL occurrence was relatively high and varied between 4 and 40%. The estimated daily intake of DON per 1 kg of body weight in Russia on average varied from 0,066 to 0,096 mg. The daily intake of DON in the North-Caucasian region was twice higher than in Russia on average and it didn't exceed tolerable daily intake (TDI) of DON - 1 mg/kg of body. HPLC-MC analyses results indicated that 14 and 16% of all investigated grain samples were contaminated by T-2 and HT-2 toxins accordingly. T-2 toxin content in grains was lower then MTL (0,1 mg/kg). High frequency and levels of fumonisins contamination were detected in the food corn.

[Fusarium toxins in the cereal crop in Russia (situation in 1993 and 1994)]. / Fuzariotoksiny v zernovykh kul'turakh v Rossii (situatsiia v 1993 i 1994 gg.).

The occurrence of deoxynivalenol (DON) and zearalenone in 495 samples of food wheat, barley and rye collected from different regions of Russia, and in 633 samples of fresh harvested wheat from Krasnodar region in 1993 and 1994 was surveyed. DON was detected in 97% of fresh harvested wheat samples in 1993, exceeding maximum tolerated level MTL) in 69% of samples analyzed. 23% of fresh harvested wheat samples were positive for DON and only in 5% of samples investigated DON concentration exceed MTL in 1994. Zearalenone was found in low concentrations in 3 of 154 wheat samples analyzed. Surveys of food wheat, barley and rye samples have shown that 23%, 7% and 3% of lots were positive for DON, respectively, in 1993. DON concentration exceed MTL in 14% of food wheat samples. The frequency of DON contamination of the 1994 food wheat samples was 6%. No mycotoxins were found in the 1994 food barley and rye samples analyzed. Almost all DON contaminated lots of food grain were collected from North Caucasus region.

[Grain contamination by deoxynivalenol in Russia]. / Problema kontaminatsii zernovykh kul'tur dezoksinivalenolom v Rossii.

Deoxynivalenol (DON) contamination of fresh harvested grain (570 samples of wheat and barley harvested in 1990-1992) and food grain crops (468 samples of wheat, rye, barley from different regions of the Russian Federation) was examined for frequency and degree. DON was detected in 70% of the fresh harvested wheat, exceeding MPC in 26% of the samples and in 67% of the stored barley samples, exceeding MTL in 14%. In 1992 100% of the harvested wheat from fusariosis natural habitat were contaminated exceeding MPC in 92%. Out of 314 samples of food wheat, 29% contained DON in quantities exceeding MTL in 14%. 11% of food barley and 8% of food rye samples contained DON within MTL. Maximum quantities of contaminated wheat were produced in the North-Caucasian regions (66% of the contaminated samples) or were imported (21%). Contaminated wheat was used most frequently in the North-Caucasian region and Central regions (56% and 30% of the contaminated samples, respectively).

[A method of determining the levels of deoxynivalenol (vomitoxin) and its metabolites]. / Metod opredeleniia dezoksinivalenola (vomitoksina) i ego metabolitov.

A method has been developed for assay of deoxynivalenol (DON) and its metabolite desepoxideoxynivalenol (DOM-1) in animal excrements with the use of reversed-phase high-performance liquid chromatography with UV-detection (218 nm). The method includes purification in the mini-column with activated carbon and aluminium oxide. The detection limit was 50 ng/g, relative standard deviation--0.05-0.1, the degree of toxin isolation--76-89%. DON isolated from Fusarium macroceras, strain 579a, cultivated in rice under laboratory conditions, and DOM-1 obtained as a result of DON incubation with the contents of the beef first stomach, were used in the study. The structure of toxins isolated has been proved by the mass-spectrometry method. The method developed by the authors was used in the study of DON metabolism in vivo in monkeys.

A survey of the occurrence of deoxynivalenol in wheat from 1986-1988 harvests in the USSR.

The occurrence of deoxynivalenol (DON; vomitoxin) in wheat from 1986-1988 harvests in the USSR was surveyed. A significant frequency of DON contamination (81.3% of samples analyzed) was observed. A correlation between levels of DON contamination and percentage of Fusarium-damaged kernels was demonstrated. It was shown that DON contamination did not exceed the maximum tolerated level (MTL) established in the USSR (1.0 mg/kg) if samples contained no more than 0.6% of Fusarium-damaged kernels.

[The contamination of the 1986-1988 wheat harvests with deoxynivalenol (vomitoxin)]. / Izuchenie zagriazneniia pshenitsy urozhaev 1986--1988 gg. dezoksinivalenolom (vomitoksinom).

The authors used high-performance liquid chromatography to estimate the content of deoxynivalenol (vomitoxin) trichothecene mycotoxin in 175 samples of wheat harvested in the Krasnodar Territory in 1986-1988. High incidence rates and levels of wheat intoxication have been recorded: 42.8% in 1986, 25% in 1987, and 80.28% of wheat samples in 1988 contained deoxynivalenol in concentrations exceeding MPC (1 mg/kg). A correlation was noted between the degree of wheat affection with Fusarium and the level of its intoxication with deoxynivalenol. A conclusion has been made on the necessity of using the criteria of the degree of wheat affection with Fusarium and the level of its intoxication with deoxynivalenol in hygienic recommendations for safe utilization of fusarial grain for food purposes.