实时荧光PCR方法检测食品中痕量荞麦成分

目的：建立一种快速、特异、灵敏的荞麦成分检测方法。 方法：针对荞麦内转录间隔区ITS(internal transcribed spacer)和5.8S rRNA基因序列设计一对PCR引物及探针,建立实时荧光PCR检测方法;以同源性(27个荞麦属相关物种)及非同源性(食品中常见的栽培型植物)参考植物物种作特异性检测;以50mg/kg小麦DNA作为稀释液对荞麦DNA进行梯度稀释,做灵敏度检测。 结果：该方法能够有效对荞麦成分进行快速检测,具有较强的特异性,灵敏度较高(可达0.1μg/kg)并且小麦成分的存在对荞麦灵敏度检测没有影响。结论：该方法特异性强,灵敏度高,可以快速、准确检测食品中含有的痕量荞麦成分。     

A quantitative competitive PCR system to detect contamination of wheat, barley or rye in gluten-free food for coeliac patients

 The only treatment in coeliac disease is a lifelong avoidance of wheat, barley and rye (WBR) in the daily nutrition. According to the Codex Alimentarius Commission of the Joint Food and Agricultural Organisation and the World Health Organisation of the United Nations, 100 ppm gliadin (10 mg gliadin/100 g dry weight) is the maximum allowed in food labelled as 'gluten-free'. The present study describes the evaluation of a quantitative competitive polymerase chain reaction (QC-PCR) system as an indication of contamination of gluten-free food with the coeliac-toxic cereals. The QC-PCR system simultaneously detects WBR-DNA on the basis of a non-coding region of chloroplast trnL gene. An internal DNA standard was constructed by adding 20-bp to the original PCR product. This standard was calibrated to 0.02% and 0.2% wheat DNA corresponding to 10 ppm and 100 ppm gliadin, respectively. The QC-PCR system was applied to 15 commercially available products labelled as 'gluten-free'. QC-PCR and ELISA yielded identical results for most cases. By QC-PCR as well as by ELISA one sample was shown to contain more than the allowed maximum limit to be labelled as 'gluten-free'. Through the application of both methods, the origin of a contamination can be identified. A positive QC-PCR signal and a negative ELISA result indicates a possible gliadin-free wheat starch addition and vice versa a possible addition of wheat-free gliadin as a food additive. Both methods support each other in testing gluten-free products. Received: 23 March 2000     

Quantification of lupine (Lupinus angustifolius) in wheat flour using real-time PCR and an internal standard material

In the European Union, labelling of the 14 food allergens listed in Annex IIIa of Directive 2000/13/EC is mandatory. The implementation of upper limits for these allergens is under discussion. Therefore, quantitative analytical methods will be needed to verify compliance with regulatory requirements and to provide an improved basis for the legal assessment of allergen labelling. In this study, the lupine flour content in wheat flours was determined using real-time PCR and statice seeds as internal standard material. The method proved to be applicable to the quantification of lupine contents from 1 to 10?mg/kg, which is in the range relevant for allergic consumers.     

Fluorinated organic compounds in an eastern Arctic marine food web

An eastern Arctic marine food web was analyzed for perfluorooctanesulfonate (PFOS, C8F17SO3-), perfluorooctanoate (PFOA, C7F15COO-), perfluorooctane sulfonamide (PFOSA, C8F17SO2NH2), and N-ethylperfluorooctane sulfonamide (N-EtPFOSA, C8F17SO2NHCH2CH3) to examine the extent of bioaccumulation. PFOS was detected in all species analyzed, and mean concentrations ranged from 0.28 +/- 0.09 ng/g (arithmetic mean +/- 1 standard error, wet wt, whole body) in clams (Mya truncata) to 20.2 +/- 3.9 ng/g (wet wt, liver) in glaucous gulls (Larus hyperboreus). PFOA was detected in approximately 40% of the samples analyzed at concentrations generally smaller than those found for PFOS; the greatest concentrations were observed in zooplankton (2.6 +/- 0.3 ng/g, wet wt). N-EtPFOSA was detected in all species except redfish with mean concentrations ranging from 0.39 +/- 0.07 ng/g (wet wt) in mixed zooplankton to 92.8 +/- 41.9 ng/g (wet wt) in Arctic cod (Boreogadus saida). This is the first report of N-EtPFOSA in Arctic biota. PFOSA was only detected in livers of beluga (Delphinapterus leucas) (20.9 +/- 7.9 ng/g, wet wt) and narwhal (Monodon monoceros) (6.2 +/- 2.3 ng/g, wet wt), suggesting that N-EtPFOSA and other PFOSA-type precursors are likely present but are being biotransformed to PFOSA. A positive linear relationship was found between PFOS concentrations (wet wt) and trophic level (TL), based on delta15N values, (r2 = 0.51, p < 0.0001) resulting in a trophic magnification factor of 3.1. TL-corrected biomagnification factor estimates for PFOS ranged from 0.4 to 9. Both results indicate that PFOS biomagnifies in the Arctic marine food web when liver concentrations of PFOS are used for seabirds and marine mammals. However, transformation of N-EtPFOSA and PFOSA and potential other perfluorinated compounds to PFOS may contribute to PFOS levels in marine mammals and may inflate estimated biomagnification values. None of the other fluorinated compounds (N-EtPFOSA, PFOSA, and PFOA) were found to have a significant relationship with TL, but BMF(TL) values of these compounds were often >1, suggesting potential for these compounds to biomagnify. The presence of perfluorinated compounds in seabirds and mammals provides evidence that trophic transfer is an important exposure route of these chemicals to Arctic biota.     

Biomagnification of alpha- and gamma-hexabromocyclododecane isomers in a Lake Ontario food web

The extent of bioaccumulation of hexabromocyclododecane (HBCD) isomers (alpha, beta, and gamma) was determined in the Lake Ontario pelagic food web using liquid chromatography tandem mass spectrometry (LC/MS/MS). Concentrations of the alpha-isomer were consistently higher than that of the gamma-isomer. The beta-isomer was below method detection limits in all samples. Whole body concentrations (ng/g, wet wt) of alpha- and gamma-HBCD were highest in the top predator lake trout samples ranging from 0.4 to 3.8 ng/g for the alpha-isomer and 0.1 to 0.8 ng/g for the gamma-isomer. For the prey fish species, the trends in alpha- and gamma-HBCD levels were slimy sculpin > smelt > alewife. Mean concentrations of total (sigma) HBCD (sum of alpha- and gamma-isomers) in the macrozooplankter Mysis relicta (0.14 +/- 0.02 ng/g wet wt) and in the benthic invertebrate Diporeia hoyi (0.16 +/- 0.02 ng/g, wet wt) were similar and approximately twice as high as in plankton (0.06 +/- 0.02 ng/g, wet wt). A strong positive linear relationship was found between sigmaHBCD concentrations (wet wt) and trophic level based on delta15N suggesting that HBCD biomagnifies in the Lake Ontario food web. The trophic magnification factor (TMF = 6.3) derived from the slope of the sigmaHBCD - trophic level relationship was slightly higher than TMFs for p,p'-DDE (6.1) and sigmaPCBs (5.7) found previously. Biomagnification factors (BMF, calculated as the ratio of lipid corrected concentration in predator/lipid corrected concentration in prey) were variable between feeding relationships and ranged from 0.4 to 10.8 for the alpha-isomer and from 0.2 to 10 for gamma-isomers.     

Development of Ephestia kuehniella (Zeller), Plodia interpunctella (Hübner) and Corcyra cephalonica (Stainton) (Lepidoptera: Pyralidae) on kernels and wholemeal flours of Fagopyrum esculentum (Moench) and Triticum aestivum L.

Three stored product moth pests, Ephestia kuehniella, Plodia interpunctella and Corcyra cephalonica were reared on the following five foods: whole buckwheat with pericarp, decorticated buckwheat, wheat var. “Centauro” (kernels), wholemeal wheat flour and whole buckwheat flour. Results showed that achenes of buckwheat with pericarp are a poor food for the development of these species. A low rate of survival to adulthood for E. kuehniella and P. interpunctella was recorded, with a considerable extension of post-embryonic development. Although the mandibles of larvae are strong, they have trouble in breaking the fibrous buckwheat pericarp. It was observed that when the seed was decorticated there was a higher percentage of adults emerged. The emergence of E. kuehniella began, according to the food given, 34–42 days after the eggs were laid. The susceptibility index (s.i.) of achenes without pericarp (s.i. 9.7) was higher than that observed on wheat (s.i. 8.6). The first P. interpunctella adults were found after 29 days on wheat and after 56 days on buckwheat with pericarp. The shortest mean period of development occurred on wheat (34 days) while the longest was on buckwheat with pericarp at 81 days. The highest susceptibility index was on kernels (s.i. 12.8), the lowest one was on buckwheat with pericarp (s.i. 2.3). C. cephalonica began to emerge, according to the food given, after 40–55 days. In this case, fewer adults were recorded from buckwheat with pericarp, but no significant differences among the means of emerged adults on wheat, wholemeal wheat flour and whole buckwheat flour were observed. The longest mean period of development was recorded on wholemeal wheat flour (72 days) while the shortest was on wheat kernels (58 days). The highest value for the susceptibility index was obtained for wheat kernels (s.i. 7.4) and the lowest one for buckwheat with pericarp (s.i. 4.5).     

Determination of unprocessed genetically modified soybean in foods using simplex and duplex real-time PCR with an internal standard

We developed a method using an internal standard to determine the amount of unprocessed genetically modified organisms (GMO) in foods as GMO weight per weight of total food (w/w_tf) using real-time PCR. Food samples were spiked with an internal standard, a ColE1 plasmid, and DNA was extracted from the samples using a silica membrane-type kit and analysed using real-time PCR. The relationship between the GMO amount and the copy number ratio of the transgene to ColE1 in 0.1–5% w/w_tf GM soybean powders was found to have a correlation coefficient ( r ) of 0.97. GMO was quantified in food samples spiked with GM soybean (final amount 0.5% w/w_tf GMO). The average GMO amount ranged from 0.35% to 0.63% w/w_tf. The results show that our method should be useful for quantifying unprocessed GMO in foods. We also developed a duplex assay, which is simpler and more accurate than the simplex assay.     

Construction and evaluation of a microbiological positive process internal control for PCR-based examination of food samples for Listeria monocytogenes and Salmonella enterica

PCR assays for food-borne pathogens are widely available but have had more limited application to food testing compared with their use in clinical laboratories. When testing food samples, false negative PCR results can occur and may be due to interference with target-cell lysis necessary for nucleic acid extraction, nucleic acid degradation and/or direct inhibition of the PCR. Therefore, it is essential to include appropriate controls for the application of PCR to the detection of pathogens in food samples. The purpose of this study was to develop and evaluate a novel internal control (IC), capable of monitoring the complete detection procedure, from DNA extraction through to amplification and detection. A 'positive process' IC was developed for the reliable application of real-time PCR assays for the detection of Salmonella enterica or Listeria monocytogenes in enrichment broths. Two novel real-time 5' nuclease PCR assays for the detection of a 77 bp fragment of the green fluorescent protein (gfp) gene and a 91 bp fragment of the iroB gene of S. enterica were developed. These assays were specific and had detection limits of 5+/-0.88 and 15+/-1.03 CFU per PCR for the gfp and iroB genes respectively. The gfp PCR assay was combined with the iroB PCR assay, and also with a previously published real-time 5' nuclease PCR assay for the detection of the hlyA gene of L. monocytogenes. Duplexed assays were optimised such that the target genes were simultaneously amplified at similar sensitivities to single reactions. The gfp gene was cloned into the chromosome of a non-pathogenic strain of Escherichia coli and the modified strain successfully encapsulated in LENTICULE discs. A single disc was added to 1 ml of standard enrichment broths immediately prior to DNA extraction, and used as an IC for the detection of L. monocytogenes and S. enterica by PCR. This method was evaluated using 393 naturally contaminated food or environmental samples, 267 for the detection of Salmonella spp. and 126 for Listeria spp. PCR inhibition was detected in 29 (8%) extracts, although neither pathogens were detected in these broths by culture. S. enterica was detected by PCR in 43 of 45 (96%) broths that were positive by conventional culture. The iroB gene was also detected in a further 2 broths by PCR alone. L. monocytogenes was detected in 6 broths by both PCR and conventional culture, plus an additional 7 by PCR only. Control strategies such as those described here are important tools for the interpretation of PCR assays by improving the reliability of detection of pathogens in food.     

Incidence and polymerase chain reaction assay of Listeria monocytogenes from raw milk in Gyeongnam Province of Korea.

A total of 50 raw milk samples from Gyeongnam Province of Korea were examined for the incidence of Listeria monocytogenes between July 1998 and August 1998. L. monocytogenes isolated by biochemical test was confirmed by polymerase chain reaction (PCR) with two sets of primers designed from the invasion-associated protein (iap) gene. After standard PCR with external primers, the amplified DNA was confirmed by a second round of PCR with internal primers (nested PCR). Both the external and internal primers generated 468-bp and 287-bp products. respectively. Only one (G9 strain) of the three suspect samples that tested positive in biochemical tests for L. monocytogenes from 50 raw milk samples was also PCR positive. Following this procedure. PCR-positive G9 strain was confirmed by Southern blot using the 287-bp internal iap probe again. The detection limit of G9 strain by standard PCR assay was as few as 102 cells, equivalent to approximately I pg of L. monocytogenes DNA. These PCR assays may be useful for novel detection as well as rapid confirmation for L. monocytogenes from food samples and the field.     

Detection of wheat contamination in oats by polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA)

 It is well established that the consumption of wheat prolamins causes the characteristic symptoms of coeliac disease (CD) in subjects who are predisposed to it. There is currently much discussion about the role of oats in the pathogenesis of CD. Evidently, it is important that oats used for clinical studies are not contaminated with wheat. In this study, 38 oat samples were investigated by polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA): 30 samples consisted of flakes or grains and 8 probes were industrially processed oat diets. Wheat prolamin (gliadin) detection by ELISA showed that 16 of these samples contained less than the detection limit of 0.2 mg gliadin/100 g dry weight; 21 samples contained less than 2.8 mg gliadin/100 g dry weight and 1 sample reached 38 mg gliadin/100 g dry weight, clearly exceeding the allowed Swiss limit of 10 mg gliadin/100 g dry weight for gluten-free products. Spiking experiments showed that the wheat PCR system is about ten times more sensitive than the ELISA system, provided that the isolated DNA is fully amplifiable. Thus, wheat DNA could be detected by the wheat PCR system in ten samples with gliadin contents below the detection limit of the ELISA system used. Applying a eukaryote-specific 18S-PCR system the presence of amplifiable DNA was verified. Only two of eight samples of industrially processed oat products contained amplifiable DNA, the other six samples had no detectable DNA left. One sample was wheat-PCR positive. However, all eight samples contained detectable amounts of gliadin in the ELISA. Received: 7 November 1997     

Quality and antioxidant property of buckwheat enhanced wheat bread

Common buckwheat (Fagopyrium esculentum Moench) was used to substitute 15% of wheat flour to make buckwheat enhanced wheat breads. Proximate composition, physical quality, functional components and antioxidant properties of buckwheat enhanced wheat breads were analysed and compared with those of white bread. Specific volumes of three breads were 6.10–6.75 cm³/g. Buckwheat enhanced wheat bread showed lower lightness and whiteness index values and higher redness and yellowness values. On a seven-point hedonic scale, all sensory results were 5.33–5.91, indicating that three breads were moderately acceptable. No differences were found in appearance, colour and overall sensory attributes for three breads, whereas both buckwheat enhanced wheat breads were rated higher in flavour and mouth feel. Buckwheat enhanced wheat bread contained more rutin and quercetin as expected. Buckwheat enhanced wheat bread was good in antioxidant activity, reducing power and 1,1-diphenyl-2-picrylhydrazyl radical scavenging ability with unhusked buckwheat enhanced wheat bread being the most effective. Overall, buckwheat enhanced wheat bread could be developed as a food with more effective antioxidant properties.     

Effect of raw wheat germ addition on the physical texture and objective color of a designer food (pan bread)

Pan bread formulations based on raw wheat germ, vital wheat gluten, and enzyme-active soybean flour were optimized with the objective of developing a phytochemical-enriched designer food product with superior nutritional and sensory qualities. The objective texture values (measured as compression force, g) indicated that the test bread with 10% wheat germ addition was comparable (299.9 g) to the control (210.1 g), but the compression force was significantly higher (415.4 g) at 20% wheat germ level. With 0.5% sodium stearoyl-2-lactylate (SSL), 30 ppm potassium bromate and 50 ppm ascorbic acid, the test breads with 10 and 20% wheat germ had compression force values of 313.8 g and 367.7 g, respectively. Comparing the CIE L*a* values, the test bread samples having up to 20% wheat germ were slightly darker in crumb color than the white flour control bread, but were significantly lighter than the whole wheat flour bread. However, the addition of wheat germ increased the yellow color of bread crumb as indicated by the higher b* values of 11.4, 16.4 and 21.4, for control, 10% and 20% wheat germ breads, respectively. The physical texture and objective color measurements can be used in evaluating the quality of a phytochemical-enriched designer food (pan bread). It can be concluded that wheat germ-enriched bread can be prepared by using white flour, 20% raw wheat germ, 0.5% SSL, 30 ppm potassium bromate and 50 ppm ascorbic acid to provide consumers with a functional food.     

Rapid detection of Listeria monocytogenes in food using culture enrichment combined with real-time PCR

A rapid method for the detection of Listeria monocytogenes in foods combining culture enrichment and real-time PCR was compared to the ISO 11290-1 standard method. The culture enrichment component of the rapid method is based on the ISO standard and includes 24 h incubation in half-Fraser broth, 4 h incubation in Fraser broth followed by DNA extraction and real-time PCR detection of the ssrA gene of L. monocytogenes. An internal amplification control, which is co-amplified with the same primers as the L. monocytogenes DNA, was also included in the assay. The method has a limit of detection of 1–5 CFU/25 g food sample and can be performed in 2 working days compared to up to 7 days for the ISO standard. A variety of food samples from retail outlets and food processing plants (n = 175) and controls (n = 31) were tested using rapid and conventional methods. The rapid method was 99.44% specific, 96.15% sensitive and 99.03% accurate when compared to the standard method. This method has the potential to be used as an alternative to the standard method for food quality assurance providing rapid detection of L. monocytogenes in food.     

Detection of wheat adulteration of spelt flour and products by PCR

 To date, no general method to detect wheat [Triticum aestivum vulgare (Vill.) Mackey] adulteration of spelt [T. aestivum spelta (L.) Thell.] exists. Exploiting a published allelic difference in γ-gliadin gene GAG56D, we developed and evaluated three PCR-based methods to determine the proportion of wheat in spelt flour and products. A sensitive wheat-specific PCR system was designed. Moreover, a heterologous internal standard was constructed for quantitative competitive (QC-)PCR. The standard was arbitrarily calibrated to 5% wheat DNA in spelt DNA. A PCR-RFLP system consisting of a GAG56D-specific PCR system with subsequent restriction was used to estimate the relative proportions of wheat and spelt in DNA mixtures. The three methods were successfully applied to seven commercial spelt samples and one self-produced bread. Five and three of the seven samples were found to contain more than 5% and 10% wheat, respectively. One sample appeared to contain no spelt at all. Analyzing the bread crumb with PCR-RFLP, we were able to estimate the wheat content of the flour which was used to produce the bread and thus demonstrated the applicability of PCR-RFLP to quantify processed products. Received: 24 March 2000     

Inter-laboratory evaluation studies for establishment of notified ELISA methods for allergic substances (buckwheat)

Inter-laboratory evaluation studies were conducted for the ELISA methods for allergic substances (buckwheat). Extracts of snack, bun and udon spiked with buckwheat standard protein at a level of 5-20 ng/mL as sample solutions were analyzed in replicate at 10 laboratories. Coefficients of variation (CVs) of the ELISA methods using a Buckwheat Protein ELISA Kit (Buckwheat kit) and a FASTKIT Buckwheat ELISA kit (Buckwheat ELISA kit) were mostly below 10%. Mean recoveries of the buckwheat standard protein from the food extracts were over 40% in the two ELISA methods. Repeatability relative standard deviations of buckwheat standard protein in three food extracts were in the ranges of 6.8-78.5% and 5.0-33.9% for the Buckwheat kit and the Buckwheat ELISA kit, respectively. Reproducibility relative standard deviations of buckwheat standard protein in three food extracts were 11.9-69.5% and 16.5-34.1% for the Buckwheat kit and the Buckwheat ELISA kit, respectively. The detection limits of both ELISA methods were 1 ng/mL in sample solutions. These results suggest that the notified ELISA methods are reliable and reproducible for the inspection of buckwheat protein levels in extracts of snack, bun and udon.     

Reduction of rutin loss in buckwheat noodles and their physicochemical characterisation

Tartary buckwheat was subjected to hydrothermal treatments for minimising rutin loss in buckwheat-based foods by water addition. When native buckwheat flour was mixed with water for 60 min, the rutin content was distinctly reduced from 3.74 g/100 g to 0.31 g/100 g, increasing the amount of quercetin. However, the rutin content remained constant and quercetin was hardly detected in hydrothermally-treated buckwheat flour. Also, when noodles were prepared with wheat and buckwheat flours (7:3, w/w), the noodle samples containing hydrothermally-treated buckwheat flour, showed higher amounts of rutin (more than 0.83 g/100 g) than the control noodle with native buckwheat flour (0.27 g/100 g). In addition, the use of hydrothermally-treated buckwheat flour gave less pasting parameters and lower viscoelastic properties. The noodle dough with hydrothermally-treated buckwheat flour also had greater water absorption and development time during mixing while the elongation stress of the noodle dough was reduced.     

适合糖尿病的几种杂粮主食面条

摘要：研究了不同配方的荞麦面条、燕麦面条和鹰嘴豆面条以及传统的小麦面条对糖尿病大白鼠餐后血糖的影响。大白鼠血糖实验结果是：荞麦面条（荞麦粉40小麦粉60）、燕麦面条（燕麦粉40小麦粉60）和鹰嘴豆面条（鹰嘴豆粉100、谷朊粉10）的血糖曲线下面积增加值分别是6.23mmol.h/l、6.56mmol.h/l和6.60mmol.h/l。其值明显低于纯小麦面条（14.07mmol.h/l）,显著性p<0.05。这表明以荞麦、燕麦和鹰嘴豆为主要原料加工而成的面条比纯小麦面条更适合作为糖尿病人的主食。     

Detection of wheat as an allergenic substance in food by a nested PCR method

A nested PCR method was developed for the detection of DNAs extracted from allergenic substances (here, wheat) in food. Because of DNA fragmentation, detection of wheat-specific DNA extracted from food, such as retort pouch food, is very difficult. Therefore, to improve the sensitivity of detection, a nested PCR primer pair (Wtr01NE2-5' and Wtr10NE5-3': amplicon size 97 bp) was newly designed within the region of the PCR products amplified by the official Japanese primer pair (Wtr01-5' and Wtr10-3'; amplicon size 141 bp) for wheat. Genomic DNAs of seven kinds of commercial processed foods containing wheat, wheat flour and three kinds of wheat flours pressure-heated at 100, 121 and 131 degrees C were extracted with a commercial ion-exchange type kit by modifying the Japanese official method. The nested PCR method involved two PCR procedures. First, PCR was performed by varying both the PCR reagents and cycling conditions of the Japanese official method. Second, PCR was performed using the first PCR products diluted 200-fold with TE buffer. The Japanese official method enabled detection of only four of the seven kinds of foods and three of the four kinds of flours (one sample was just a trace), while the nested PCR method detected all seven foods and all four flours. Investigation of the detectability of the four kinds of wheat flours depending on the size of the amplified fragment using five primer pairs showed that its size must be kept to less than approximately 100 bp. The nested PCR method significantly improved the sensitivity of detection of wheat-specific DNA.     

可可粉中常见谷物成分的PCR检测技术研究

实验利用PCR方法检测可能掺入可可粉中的谷物成分。通过比较6种可可粉DNA的提取方法,建立了一种可靠、快速的方法,提出的DNA能够用于后续PCR反应。同时设计并验证了谷物通用引物的特异性和通用性,包括水稻、小麦、玉米、高粱米、荞麦、燕麦等常见谷物可通过1次PCR扩增筛选出来。模拟可可粉样品中谷物成分检出限达到0.1%。使用该方法检测了市场上购买的14个样品,均未检出常见谷物成分。为可可粉中可能添加的外源性物种检测提供了快速有效检测的方法和思路。     

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 10(2) spores after 16 h of incubation.