Minaeva L P, Korotkevich Iu V, Sheveleva S A
Vopr Pitan. 2013;82(3):61-6.
Currently contamination of food grains with Fusarium spp. is determined by conventional mycological methods and can last up to 30-40 days. The method specificity is highly dependent on the subjective evaluation of the researchers. The alternative to traditional mycological methods is detection by PCR. The purpose of the study was to improve the contamination analysis method of food grains infected by Fusarium for analysis time reducing and species detection specificity increasing. Investigations were carried out on food grains samples harvested in 2009-2011 from 5 federal regions of Russia. On the first stage, 100 grains were sowing on potato-sucrose medium and then incubated at 24 degrees C for 7-10 days. At the second stage, the Fusarium species composition grown from food grains was estimated by two methods. The first method was mycological for monospore isolates. The second one was PCR analysis of DNA extracts from the combined sample of Fusarium mycelium which grew on a foodgrains sample. Species-specific primers of such mycotoxins producers as F. graminearum, F. culmorum, F. sporotrchiodes, F. langsethiae, F. poae, F. avenaceum, F. tricinctum were used for PCR detection. The species composition of viable Fusarium spp. fungi revealed in foodgrains samples by mycological method completely concided with the results of PCR analysis. In result, the method that combines traditional mycological sowing for detection of viable species with PCR species detection of the mycelium in integrated sample has been developed. The method significantly reduces test duration (3-4 times) by excluding the sieving step in obtaining monospore fungi isolates for further species identification. The method also allows obtaining reliable data on the viable Fusarium species including producers of toxins in foodgrains. Thus, the idea of improving of the identification stage allowing reducing labor costs and increasing of the method specificity was realized.
目前，谷物被镰刀菌属污染是通过传统的真菌学方法来测定的，整个过程可能持续30至40天。该方法的特异性高度依赖于研究人员的主观评估。传统真菌学方法的替代方法是通过聚合酶链反应（PCR）进行检测。本研究的目的是改进受镰刀菌感染的谷物污染分析方法，以缩短分析时间并提高物种检测特异性。研究针对2009年至2011年从俄罗斯5个联邦地区采集的谷物样本展开。第一阶段，将100粒谷物播种在马铃薯蔗糖培养基上，然后在24摄氏度下培养7至10天。第二阶段，通过两种方法评估从谷物中生长出来的镰刀菌物种组成。第一种方法是对单孢子分离物进行真菌学分析。第二种方法是对在谷物样本上生长的镰刀菌菌丝体混合样本的DNA提取物进行PCR分析。使用了禾谷镰刀菌、燕麦镰刀菌、拟枝孢镰刀菌、兰氏镰刀菌、梨孢镰刀菌、燕麦镰孢菌、三线镰刀菌等产真菌毒素物种的特异性引物进行PCR检测。通过真菌学方法在谷物样本中检测到的存活镰刀菌属真菌的物种组成与PCR分析结果完全一致。结果，开发出了一种将用于检测存活物种的传统真菌学播种方法与对综合样本中的菌丝体进行PCR物种检测相结合的方法。该方法通过省去获取单孢子真菌分离物以进行进一步物种鉴定的筛选步骤，显著缩短了检测时间（缩短了3至4倍）。该方法还能够获取关于谷物中包括毒素产生菌在内的存活镰刀菌物种的可靠数据。因此，实现了改进鉴定阶段以降低劳动力成本并提高方法特异性的想法。
