To validate commercial kit for screening of the presence of AFB1 in corn, an interlaboratory study was conducted to evaluate three quantitative and two qualitative immunoassay kits designed for the detection of aflatoxins. Four laboratories performed a screen for the presence of aflatoxin B1 (AFB1) in corn. As for the quantitative kits, repeatability relative standard deviation (RSDr) and reproducibility relative standard deviation (RSDR) were estimated. One laboratory evaluated the lot variation of each quantitative kit. All kits used in this study showed that the RSDr and RSDR were less than 23.3 % and 35.7 %, respectively, in spiked or naturally contaminated corn samples. As for the qualitative kits, neither false positive nor false negative results were found in corn samples (blank, spiked or naturally contaminated samples) in any laboratories. The RSDs in the lot variation of the same quantitative kit was less than 46.5 % in both two brands. The results appeared to indicate that all kits tested in this study could be validated for the screening of the presence of AFB1 in corn, and were also available for the first step of the detection of AFB1 at levels of more than 5 ng/g.
Ochratoxin A (OA) is a mycotoxin produced by certain Aspergillus and Penicillium fungi. Contamination with OA can occur in many foods such as cereals, coffee, wine, dried fruits, and meat products. However, little is known about ochratoxin contamination on rice, which is a staple food for many Asian countries. In this study we examined a method of analysis for OA in rice and buckwheat. Five types of samples, polished japonica type rice (short grain), polished glutinous rice, rough japonica type rice, rough indica type rice (long grain) and buckwheat (two samples) were tested. Samples were spiked with OA over the range from 0.1 to 10.0 μg/kg for the linearity study and at 0.5 and 5.0 μg/kg for the reproducibility study. Ochratoxin A was extracted with acetonitrile/water, isolated using an immunoaffinity column, separated by HPLC, and detected by fluorescence. At 0.5 μg/kg level, within day recoveries were 64.5 to 110.2 % and Relative Standard Deviations (RSDs) were 5.7 to 16.7 % and between days, recoveries were 55.8 to 99.6 % and RSDs were 4.3 to 15.0 %. Average recoveries of OA in the range of 0.2 to 10 μg/kg were 89.6 to 118.1 %. From these result, it is shown that this method can be applied for the analysis of OA present as a contaminant in rice and buckwheat in the range from 0.2 to 10 μg/kg.
In this study, we performed the side population (SP) cell analysis and the expression analysis of stemness genes to identify stem-like cells (cancer stem cells) in aflatoxin B1-(AFB1) induced rat hepatoma K2 cells. Using the flow cytometric analysis with rhodamine 123, the SP cells, which are thought to be cancer stem cells, were detected with high frequency and estimated to be approximately 89 % of K2 cell population. Furthermore, K2 cells expressed stemness genes including the sox2, nanog, klf4, the ras family E-ras and the polycomb family bmi1 genes, which are implicated in the maintenance of pluripotency and self-renewal of embryonic stem (ES) cells. On the other hand, in K2 cells the expression levels of mature hepatocyte-specific marker genes such as albumin and tyrosine aminotransferase (TAT) were very low. Thus, these results imply that K2 cells possess the typical properties of cancer stem cells through the acquisition of stemness gene expressions.
Rice is susceptible to the fungus Gibberella fujikuroi, the anamorph of which is taxonomically near to Fusarium verticillioides, a major fumonisin producer, so that the potential risk of fumonisin contamination in rice is significant. However, most works on fumonisins have examined corn or wheat and there have not been any practical methods developed for the analysis of fumonisins in rice. This paper describes the development of liquid chromatographic determination methods with an efficient extraction procedure for fumonisins (fumonisin B1, B2, and B3) in rice, using artificially molded rice contaminated with fumonisins. Applied with submergence extraction, stable and sufficient recovery was achieved for various rice samples in spike and recovery tests. The determination was done by liquid chromatography using fluorescence detection (HPLC-FL) with precolumn derivatization, or using tandem mass spectrometry (LC-MS/MS). The limit of detection achieved by LC-MS/MS was ten times lower than that by HPLC-FL for fumonisin B1.
Method validation is one of the key activity to assure the analytical data and method have to be validate and verified before use. For method validation, harmonized collagorative validation (HCV) is most prestegious method of validation but it is not necessary to validate all methods by HCV. As method has its fit for purpose, method of validation has its fit for purpose. About method validation, AOACInternational leads the world but these days there are many issues about method validation itself. However, use of validated method with verification is the starting and one of key for laboratory quality assuarance.
Total 7933 wheat, barley and rye samples collected from major cultivation areas in Japan were examined for deoxynivalenol (DON) contamination in 2002 - 2006. From about a half of these samples, more than 0.05 mg/kg of DON was detected. More than 1.1 mg/kg of DON, which is the provisional limit regulated by the Ministry of Health, Labour and Welfare in Japan was detected from 7.8 % of the samples in 2002, 4.0 % of the samples in 2003, 2.5 % of the samples in 2004, 0.2 % of the samples in 2005 and 2.8 % of the samples in 2006.
Regulations of mycotoxins has been established for aflatoxinB1, zearalenone and deoxynivalenol in feed in Japan. We conducted analysis of mycotoxins in feed in accordance with the official methods of feed analysis in Japan. As for the contamination rates of aflatoxinB1 in maize and formula feed, the differences for every year were observed. Meanwhile, as for feed ingredients, aflatoxins were detected in what were mainly imported from Southeast Asia. In addition, we have surveyed contamination of feed ingredients and formula feeds by other mycotoxins such as ochratoxinA, deoxynivalenol, nivalenol, zearalenone and fumonisinB1.
Use of appropriate fungicides is most effective and reliable practice to control Fusarium head blight (FHB). Therefore, re-evaluation of registered fungicides and screening new candidates for control of mycotoxin contamination are considered mandatory. We found that seven chemicals; metoconazole, tebuconazole, captan, thiofanate-methyl, oxine-copper, copper-hydroxide and phosphorous acid would control deoxynivalenol (DON) and nivalenol (NIV). On the contrary, treatment of azoxystrobin significantly increased the level of DON and NIV compared to the control plots, even though it reduced disease severity of FHB. We propose that new fungicide evaluation system based on efficacy for mycotoxin contamination should be introduced. In order to develop effective strategies for controlling the risk of toxin contamination, we investigated the manner in which toxin accumulation occurs in grain. The results indicated (i) high levels of DON and NIV can be produced beyond 20 days after anthesis (DAA) even by early infection, and (ii) infection at a late stage, at least as late as 20 DAA, can cause grain contamination with these toxins. Thus, developing control strategies that cover the late stage as well as the early stage would be desirable to reduce the risk of toxin contamination in wheat.