Highly sensitive PCR-based detection method specific for Aspergillus flavus in wheat flour

Aspergillus flavus is frequently found in food, producing a wide variety of toxins, aflatoxins being the most relevant in food safety. A specific PCR-based protocol for this species is described which allowed discrimination from other closely related species having different profiles of secondary metabolites from the Aspergillus Section Flavi, particularly A. parasiticus. The specific primers were designed on the multi-copy internal transcribed region of the rDNA unit (ITS1-5.8S-ITS2 rDNA) and were tested in a wide sample of related species and other fungal species commonly found in food. The PCR assay was coupled with a fungal enrichment and a DNA extraction method for wheat flour to enhance the sensitivity of the diagnostic protocol. The results indicated that the critical PCR amplification product was clearly observed for wheat flour contaminated by 10² spores after 16 h of incubation.     

Specific detection of Aspergillus parasiticus in wheat flour using a highly sensitive PCR assay

Aspergillus parasiticus is one of the most important aflatoxin-producing species that contaminates foodstuffs and beverages for human consumption. In this work, a specific and highly sensitive PCR protocol was developed to detect A. parasiticus using primers designed on the multicopy internal transcribed region of the rDNA unit (ITS1-5.8S-ITS2 rDNA). The assay proved to be highly specific for A. parasiticus when tested on a wide range of related and other fungal species commonly found in commodities, and allowing discrimination from the closely related A. flavus. Accuracy of detection and quantification by conventional PCR were tested with genomic DNA obtained from wheat flour artificially contaminated with spore suspensions of known concentrations. Spore concentrations equal or higher than 10⁶ spore/g could be detected by the assay directly without prior incubation of the samples. The assay described is suitable for incorporation in routine analyses at critical points of the food chain within HACCP strategies.     

Limits of a PCR-based detection method for genetically modified soya beans in wheat bread production

 The official PCR-based method for the detection of recombinant DNA from glyphosate-tolerant soya beans (GTS), laid down in the collection of methods according to Sect. 35 of the German Food Law, was investigated for applicability. As a model, wheat bread was produced with a 1% addition of baking aid consisting of 45% GTS flour. DNA extraction of samples drawn at various stages of the production process revealed that during the process a degradation of DNA took place, resulting in fragment sizes in bread of <500 bp. GTS DNA was detectable at all stages, although the content of GTS flour in the dough and bread had dropped to only 0.4%. In 2 out of 15 commercial baking aids, GTS DNA was detected, reflecting the status of the use of GTS in that area. A model was also developed to study the effect on the detection of GTS under conditions when the target gene sequence of one primer is present in food originating from natural contamination or genetically modified organisms other than GTS. It was observed that high concentrations of competing DNA inhibited the PCR. This inhibition was overcome by increasing the concentration of Taq polymerase in the reaction mixture. Received: 30 April 1998 / Revised version: 17 June 1998     

A real-time PCR method for the detection and quantification of lupin flour in wheat flour-based matrices

Lupin flour is growingly being used in bakery products, mainly as a soybean protein substitute. The aim of the present work was to detect and quantify the presence of lupin flour in wheat-based foods using a newly set up qPCR system based on SYBR green. Although DNA sequence information for lupin is scarce, it has been possible to design a primer pair highly specific for the target gene and devoid of any primer-dimers amplification capacity. Lupin flour revealed to be a difficult matrix, since large amounts of compounds tend to co-purify with DNA, even adopting well established extraction protocols. Nonetheless, the primers used allowed to reach high PCR efficiencies and did not show any cross-reactivity with DNAs extracted from various plant and animal foods. The sensitivity achieved was 7 pg of lupin DNA, corresponding to a percentage of less than 0.1% of lupin flour in the foods.     

利用扫描电子显微镜快速检测大米中的黄曲霉菌

以大米作为研究对象,采用JSM‐6490LV型扫描电子显微镜检测大米中的黄曲霉菌。通过观察可以发现,正常大米胚坑干净,表面光滑,无菌丝;微黄大米胚坑内菌丝丰富,形成孢子,表面发现少量菌丝污染;微绿大米胚坑内充满菌丝和孢子,传播严重,表面被大量菌丝污染。检测加上预处理全过程耗时约20 min ,与传统方法相比,大大缩短了检测时间,可以满足现场快速检测的要求。     

A rapid method for the determination of pentosans in wheat flour

A rapid and reproducible method is presented for the determination of pentosans in wheat flour.Flour (approximately 5 mg) is added to 2ml of water followed by 10ml of a solution of glacial acetic acid, hydrochloric acid, phloroglucinol and glucose in a stoppered boiling tube.The boiling tube is placed in a water bath for twenty-five minutes and the absorbance of the resulting solution measured at 552nm and 510nm.The absorbance of interfering sugars differs very little at these two wavelengths but is considerable for the pentosans. Accordingly, the pentosans may be estimated, even when an excess of interfering sugars is present, by subtracting the absorbance at 510nm from that at 552nm.     

PCR-restriction fragment length analysis of aflR gene for differentiation and detection of Aspergillus flavus and Aspergillus parasiticus in maize

Contamination of food and feedstuffs by Aspergillus species and their toxic metabolites is a serious problem as they have adverse effects on human and animal health. Hence, food contamination monitoring is an important activity, which gives information on the level and type of contamination. A PCR-based method of detection of Aspergillus species was developed in spiked samples of sterile maize flour. Gene-specific primers were designed to target aflR gene, and restriction fragment length polymorphism (RFLP) of the PCR product was done to differentiate Aspergillus flavus and Aspergillus parasiticus. Sterile maize flour was inoculated separately with A. flavus and A. parasiticus, each at several spore concentrations. Positive results were obtained only after 12-h incubation in enriched media, with extracts of maize inoculated with A. flavus (101 spores/g) and A. parasiticus (104 spores/g). PCR products were subjected to restriction endonuclease (HincII and PvuII) analysis to look for RFLPs. PCR-RFLP patterns obtained with these two enzymes showed enough differences to distinguish A. flavus and A. parasiticus. This approach of differentiating these two species would be simpler, less costly and quicker than conventional sequencing of PCR products.     

小麦粉中溴酸钾检测方法的比较

为了选择一种最适合小麦粉中溴酸钾的检测方法,分别对4种检测方法(滴定法、反滴定法、离子色谱法和液相色谱法)进行探讨,比较它们的原理、方法、精确度、检出限、回收率及稳定性等方面。结果表明,离子色谱法操作简单,灵敏度高,溴酸根离子的最低检出浓度为50μg/kg,标准曲线具有良好的线性关系,回收率达到92.5%,是溴酸钾检测定量的较好方法,适合广泛推广和应用。     

小麦粉中磷酸盐本底含量检测研究

小麦粉是日常生活和许多食品加工行业常用的食品原料,而磷酸盐在其加工制品生产加工过程中起着重要的膨松、抗氧化等作用,国标要求用GB/T 5009.87 - 2003《食品中磷的测定》第三法对小麦粉及其加工制品进行磷酸盐含量检测,但小麦粉的磷酸盐本底含量对小麦粉加工制品磷酸盐检测结果的影响,目前国内外未有相关报道.为了确认国标法测定市售小麦粉磷酸盐含量时,小麦粉本底对结果的影响,测定了华北、东北地区10个自磨小麦粉磷酸盐含量,结果显示自磨小麦粉的磷酸盐检测值达到了2.55g/kg～3.59g/kg,此检测值已经接近国家卫生标准规定的磷酸盐限量值5g/kg.市售小麦粉及其制品的磷酸盐检测量实则为小麦粉本底磷酸盐含量及磷酸盐添加剂的总量.因此在小麦粉磷酸盐检测时有必要考虑本底值的影响,以上检测结果为小麦粉生产加工过程中磷酸盐添加限量值提供科学数据.     

A PCR-based method for the detection of Streptococcus agalactiae in milk

Bovine mastitis caused by Streptococcus agalactiae is mainly subclinical and therefore can be diagnosed only in the laboratory. We developed a polymerase chain reaction (PCR)-based method for specific and sensitive detection of S. agalactiae in raw milk. The specificity of the PCR reaction is based on unique S. agalactiae DNA sequences within the 16S subunit of the rRNA genes. Two pairs of sequences were used as positive controls; general streptococci primers, which anneal to conserved areas within the 16S rRNA subunit gene, and primers, which anneal to sequences within bovine mitochondrial DNA. The method of detection includes selective enrichment of S. agalactiae in the milk sample, followed by DNA extraction using a rapid and simple procedure developed for this purpose, and specific PCR reaction with appropriate controls. The method enables the detection of one bacterium in 1 ml of raw milk. The method developed can be easily incorporated as part of routine screening of bulk milk collection tanks for early detection of infected cows in a herd.     

Interlaboratory diagnostic accuracy of a Salmonella specific PCR-based method

A collaborative study involving four European laboratories was conducted to investigate the diagnostic accuracy of a Salmonella specific PCR-based method, which was evaluated within the European FOOD-PCR project (http://www.pcr.dk). Each laboratory analysed by the PCR a set of independent obtained presumably naturally contaminated samples and compared the results with the microbiological culture method. The PCR-based method comprised a preenrichment step in buffered peptone water followed by a thermal cell lysis using a closed tube resin-based method. Artificially contaminated minced beef and whole broiler carcass-rinse resulted in a detection limit of less than 5 cells per 25 g meat or 100 ml broiler rinse. A total of 435 samples from four countries, including pig carcass swabs (n = 285), whole broiler carcass-rinse (n = 25), various raw meat (n = 33), and environmental samples (n = 92) were investigated. The interlaboratory diagnostic accuracy, i.e. diagnostic specificity and sensitivity, was shown to be 97.5%. The co-amplification of an internal amplification control indicated possible inhibitory substances derived from the sample. This work can contribute to the quality assurance of PCR-based diagnostic methods and is currently proposed as international standard document.     

基于计算机色度学小麦粉中硼砂的快速检测方法研究

根据硼砂在酸性条件下转化为硼酸,硼酸与姜黄素显色的原理,利用自主设计的图像采集传感器,采集硼砂与显色液显色后的图像,用Lab VIEW设计图像处理软件,建立了基于计算机色度学小麦粉中硼砂的快速筛查方法。优化了显色体系的显色条件和软件的操作功能。结果表明:显色体系的亮度I值与硼酸在0~4 mg/L范围内呈线性关系,并得到测定的硼砂含量与I值的关系曲线。将关系曲线公式植入数据分析系统,通过采集显色后溶液的图像,软件可自动得出硼砂的实际添加量。该法用于实际样品分析,加标回收率为96.6%~102.9%,相对标准偏差(RSD)为1.18%~3.14%。     

基于太赫兹技术的小麦粉品质快速检测研究

该研究针对目前小麦粉品质方面检测方法存在的问题,提出利用太赫兹光谱技术对小麦粉进行快速无损品质检测研究。使用光谱仪与成像仪,采集了不同种类小麦粉样本的太赫兹光谱,使用TQ Analyst软件结合距离匹配法对小麦粉的太赫兹扫描光谱进行定性分析研究,富强粉和麦芯粉成功分类,模型性能指数达到88.9%,预测准确率达100%。使用OPUS软件结合偏最小二乘法（PLS）和一阶导数+矢量归一化（SNV）进行定量分析研究,水分定量模型R2为91.18%,交叉验证均方根为0.182;灰分定量模型 R2为83.37%,交叉验证均方根为0.064,最终通过实验结果分析得出太赫兹技术在食品品质检测方面的可行性。     

小麦比重实验研究

小麦比重实验的2个关键参数是小麦试样质量和测试用酒精体积。针对我国5个地区的小麦,研究不同试样质量和酒精体积对小麦比重的影响。结果表明,随着小麦试样质量的增加,小麦比重逐渐增加,最终趋于稳定;酒精体积对比重影响不大。现有小麦比重实验标准所采用的试样质量偏少,导致小麦比重测定结果偏低。对比研究表明,小麦比重实验的试样质量应不少于100 g,远大于现有标准推荐的10 g。     

小麦粉中过氧化苯甲酰含量的简易判断方法

提出了快速测定小麦粉中过氧化苯甲酰的方法,根据小麦粉提取液色泽的深浅与其过氧化苯甲酰含量之间的相关性规律,通过肉眼观察提取液色泽的深浅即可快速判断小麦粉中过氧化苯甲酰的大致含量。     

Evaluation of a rapid PCR-based method for the detection of animal material

A rapid PCR-based analytical method for detection of animal-derived materials in complete feed was developed. Using a commercially available DNA forensic kit for the extraction of DNA from animal feed, a sensitive method was developed that was capable of detecting as little as 0.03% bovine meat and bone meal in complete feed in under 8 h of total assay time. The reduction in assay time was accomplished by reducing the DNA extraction time to 2 h and using the simpler cleanup procedure of the kit. Assay sensitivity can be increased to 0.006% by increasing the DNA extraction time to an overnight incubation of approximately 16 h. Examination of dairy feed samples containing either bovine meat and bone meal, porcine meat and bone meal, or lamb meal at a level of 0.1% (wt/wt basis) suggested that this method may be suitable for regulatory uses. The adoption of this commercially available kit for use with animal feeds yields an assay that is quicker and simpler to perform than a previously validated assay for the detection of animal proteins in animal feed.     

含水量对玉米粉贮藏期黄曲霉生长及黄曲霉毒素B1积累的影响

目的 研究含水量对玉米粉贮藏过程中黄曲霉生长及黄曲霉毒素B1积累的影响。方法 将初始水分含量10.42%、13.26%、16.35%、19.88%的玉米粉在28 ℃, 相对湿度75%的模拟环境中储藏30 d; 测定贮藏期间玉米粉含水量、黄曲霉生长量及黄曲霉毒素B1的积累量。结果 贮藏期间玉米粉中含水量呈现先上升后下降的变化趋势, 黄曲霉生长量及黄曲霉毒素B1的积累量随贮藏时间的延迟先快速升高, 后趋于平稳。初始含水量较高的玉米粉贮藏期间含水量、黄曲霉生长量及黄曲霉毒素B1含量始终处于较高的水平。相关性分析表明, 玉米粉含水量、黄曲霉生长量和毒素积累量之间均呈极显著的正相关(P<0.01)。结论 含水量是影响玉米粉贮藏过程中霉菌生长和毒素积累的关键因素, 降低玉米粉初始含水量可以有效减轻贮藏期间真菌危害。     

The impact of fullerenol nanoparticles on the growth of toxigenic Aspergillus flavus and aflatoxins production in vitro and in corn flour

Antifungal and antimycotoxigenic activity of fullerenol nanoparticles (FNPs) were investigated on Aspergillus flavus growth isolated from a real food sample and aflatoxins (AFs) (AFB₁ and AFB₂) production. The final FNPs concentrations in in vitro and in commercial corn flour after the stationary incubation period of 7 and 14 days were in the range 0.16–80 µg/mL and 0.16–80 µg/g, respectively. Nanocharacterization of FNPs revealed an average size of 5–20 nm and a zeta potential of −35 mV. The highest degree of A. flavus mycelium growth inhibition (28%) after 7 days was observed for applied FNP concentration of 8.0 µg/mL, while after 14 days FNP concentration of 0.32 µg/mL led to the maximal inhibition of A. flavus mycelium growth (36%). Spearman's correlations analysis revealed a strong positive correlation between AFB₁ and AFB₂ concentrations in YES broth after 7 (R = 0.994, p < 0.05) and 14 days (R = 0.976), as well as between AFs concentrations and A. flavus mycelium mass after 7 (R = 0.786 for AFB₁ and R = 0.766 for AFB₂) and 14 days (R = 0.810 for AFB₁ and R = 0.833 for AFB₂). Paired samples t-test showed the existence of a statistically significant difference (p < 0.05) between the produced AFs concentrations after the incubation of 7 and 14 days. Regarding the artificially inoculated corn flour the lower applied FNP concentrations (0.16–0.8 µg/g) achieved a reduction of AFB₁ up to 42% and 60% after 7 and 14 days, respectively.