Specific detection of potentially allergenic peach and apple in foods using polymerase chain reaction

Two PCR methods were developed for specific detection of the trnS-trnG intergenic spacer region of Prunus persica (peach) and the internal transcribed spacer region of Malus domestica (apple). The peach PCR amplified a target-size product from the DNA of 6 P. persica cultivars including 2 nectarine and 1 flat peach cultivar, but not from those of 36 nontarget species including 6 Prunus and 5 other Rosaceae species. The apple PCR amplified a target-size product from the DNA of 5 M. domestica cultivars, but not from those of 41 nontarget species including 7 Maloideae and 9 other Rosaceae species. Both methods detected the target DNA from strawberry jam and cookies spiked with peach and apple at a level equivalent to about 10 μg of total soluble proteins of peach or apple per gram of incurred food. The specificity and sensitivity were considered to be sufficient for the detection of trace amounts of peach or apple contamination in processed foods.     

Differential detection of shrimp and crab for food labeling using polymerase chain reaction

Shrimp and crab are well-known as allergenic ingredients. According to Japanese food allergy labeling regulations, shrimp species (including prawns, crayfishes, and lobsters) and crab species must be differentially declared when ≥10 ppm (total protein) of an allergenic ingredient is present. However, the commercial ELISA tests for the detection of crustacean proteins cannot differentiate between shrimp and crab. Therefore, two methods were developed to discriminate shrimp and crab: a shrimp-PCR method with postamplification digestion and a crab-PCR method that specifically amplifies a fragment of the 16S rRNA gene. The sensitivity and specificity of both PCR methods were verified by experiments using DNA extracted from 15 shrimp species, 13 crab species, krill, mysid, mantis shrimp, other food samples (cephalopod, shellfish, and fish), incurred foods, and commercial food products. Both PCR methods could detect 5 pg of DNA extracted from target species and 50 ng of genomic DNA extracted from incurred foods containing 10 ppm (μg/g) total protein of shrimp or crab. The two PCR methods were considered to be specific enough to separately detect species belonging to shrimp and crab. Although false-positive and false-negative results were obtained from some nontarget crustacean species, the proposed PCR methods, when used in conjunction with ELISA tests, would be a useful tool for confirmation of the validity of food allergy labeling and management of processed food safety for allergic patients.     

New method of DNA isolation from two food additives suitable for authentication in polymerase chain reaction assays

Locust bean gum and guar gum are galactomannans used as additives (E 410 and E 412, respectively) in the food industry as stabilizing agents. Analytical discrimination between the two additives in gums and foods is now feasible by molecular techniques. However, only complex and time-consuming DNA isolation protocols are available to date. We have developed simple improved protocols to obtain enough DNA suitable for PCR amplification from a few milligrams of commercial E 410 and E 412 additives (containing more than 75% polysaccharides). The suspension of additives in water or 10 mM Tris-HCl, pH 8.5, efficiently recovers DNA suitable for authentication in PCR assays. However, the Tris method was much more efficient for the extraction of DNA from E 410 than for E 412 additives. Conversely, the water method was the most suitable for detecting DNA extracted from E 412 or from E 410/E 412 mixtures. Combined with the use of the two specific ribosomal primer pairs previously designed, our methods are well-suited for a fast and simple high-throughput sample treatment of commercial gums for molecular certification.     

Detection of recombinant DNA segments introduced to genetically modified maize (Zea mays)

Polymerase Chain Reaction (PCR) techniques are increasingly used for the detection of genetically modified (GM) crops in foods. In this paper, recombinant DNAs introduced into the seven lines of GM maize, such as Event 176, Bt11, T25, MON810, GA21, DLL25, and MON802, are sequenced. On the basis of the obtained sequence, 14 primer pairs for the detection of the segments, such as promoter, terminator regions, and construct genes, were designed. To confirm the specificities of the designed primer pairs, PCR was performed on genomic DNAs extracted from GM and non-GM maize, GM and non-GM soy, and other cereal crops. Because the presence of the corresponding DNA segments was specifically detected in GM crops by the designed primer pairs, it was concluded that this method is useful for fast and easy screening of GM crops including unauthorized ones.     

Development of a real-time PCR and a sandwich ELISA for detection of potentially allergenic trace amounts of peanut (Arachis hypogaea) in processed foods

Hidden allergens in food products are, especially for peanut-allergic consumers, a serious problem because even low amounts (approximately 200 microg) of peanut can elicit allergic reactions. Undeclared peanut traces can be found in processed food products, because contaminations with peanut during production processes are frequent. To minimize the risk of such cross-contaminations, it is necessary to develop sensitive analytical methods for the detection of hidden allergens in foods. For this approach we developed two peanut-specific assays based on the detection of peanut protein by specific antibodies (sandwich ELISA) and by the detection of peanut-specific DNA (part of the coding region of Ara h 2) by a real-time PCR. Both tests did not show any cross-reactivity with 22 common food ingredients (cereals, nuts, legumes), and the limit of detection is <10 ppm peanut in processed foods. Thirty-three random samples of food products were tested for the presence of peanut to compare both assay types with each other and to evaluate the percentage of foods on the German market that are contaminated with peanut traces. We found that four products (13.3%) without peanut in the list of ingredients contained peanut protein in a range from 1 to 74 ppm peanut protein and that the results of both tests correlated well. The real-time PCR was able to detect one more positive sample than the sandwich ELISA. In conclusion, both assays are sensitive and specific tools for the detection of hidden allergens in processed foods.     

人参EST-SSR标记的建立

从7055条人参EST序列中共搜索出791个SSR。根据引物设计标准,设计了68对EST-SSR引物。在合适的PCR反应体系下,分别以人参品种集安长脖、抚松二马牙的DNA为模板,对设计的EST-SSR引物进行筛选,发现有43对EST-SSR引物能扩增出产物。在9个人参品种、2个西洋参品种和2个刺五加品种中进一步对这些可扩增的引物对进行多态性检测,发现有26对引物显示多态性,占可扩增引物的60.47%,占设计引物总数的38.23%。本研究结果表明,根据人参EST建立EST-SSR标记是一种行而有效的的方法。     

Quantitative real-time PCR effectively detects and quantifies colonization of sclerotia of Sclerotinia sclerotiorum by Trichoderma spp.

Some members of the fungal genus Trichoderma are able to colonize and destroy sclerotia, the thick-walled resting structures of the soilborne plant pathogenic fungus Sclerotinia sclerotiorum, thus providing a potential means of biological disease control. However, current methods to detect and quantify colonization of sclerotia are labor-intensive, and generally qualitative rather than quantitative in nature. Our objective was to develop quantitative real-time PCR (polymerase chain reaction) methods to detect and measure colonization of sclerotia by Trichoderma spp. Specific PCR primer/probe sets were developed for Trichoderma spp. and for S. sclerotiorum. A total of 180 ITS1 (internal transcribed spacer) and ITS2 sequences from different species in the genus Trichoderma were aligned, and consensus sequences were determined. Six candidate primer sets were based on conserved regions of the consensus sequence, and the specificity of each nucleotide sequence was examined using BLAST (Basic Local Alignment Search Tool; NCBI) software. Each candidate primer set was tested on genomic DNA of T. harzianum strain ThzID1-M3, as well as six different Trichoderma isolates from soil, and on genomic DNA of S. sclerotiorum. The optimum primer/probe set selected, TGP4, successfully amplified genomic DNA of all Trichoderma isolates tested, and showed high precision and reproducibility over a linear range of eight orders of magnitude, from 85 ng to 8.5 fg of T. harzianum genomic DNA. TGP4 did not amplify S. sclerotiorum DNA. A specific PCR primer/probe set (TMSCL2) was developed for S. sclerotiorum, based on the calmodulin gene sequence. TMSCL2 did not amplify Trichoderma DNA. Quantitative real-time PCR with the primers then was evaluated in experiments to test differential effects of two soil moisture levels (−50 kPa, −1000 kPa matric potential) on biocontrol of S. sclerotiorum by indigenous Trichoderma spp. Periodically over 40 days, Trichoderma and S. sclerotiorum DNA in sclerotia were quantified by PCR with appropriate primers. Over 90% of the sclerotia were colonized by indigenous Trichoderma spp. at −1000 kPa, over the 40-day period, compared to only 60% at −50 kPa. In addition to determining incidence of colonization, the PCR method allowed measurement of the extent of sclerotial colonization, which also was significantly greater in the drier soil. Quantitative real-time PCR with the TGP4 primer/probe set provides a sensitive detection and measurement tool to evaluate colonization of sclerotia by Trichoderma spp.     

Comparative studies of the quantification of genetically modified organisms in foods processed from maize and soy using trial producing

Seven types of processed foods, namely, cornstarch, cornmeal, corn puffs, corn chips, tofu, soy milk, and boiled beans, were trial produced from 1 and 5% (w/w) genetically modified (GM) mixed raw materials. In this report, insect resistant maize (MON810) and herbicide tolerant soy (Roundup Ready soy, 40-3-2) were used as representatives of GM maize and soy, respectively. Deoxyribonucleic acid (DNA) was extracted from the raw materials and the trial-produced processed food using two types of methods, i.e., the silica membrane method and the anion exchange method. The GM% values of these samples were quantified, and the significant differences between the raw materials and the trial-produced processed foods were statistically confirmed. There were some significant differences in the comparisons of all processed foods. However, our quantitative methods could be applied as a screening assay to tofu and soy milk because the differences in GM% between the trial-produced processed foods and their raw materials were lower than 13 and 23%, respectively. In addition, when quantitating with two primer pairs (SSIIb 3, 114 bp; SSIIb 4, 83 bp for maize and Le1n02, 118 bp; Le1n03, 89 bp for soy), which were targeted within the same taxon specific DNA sequence with different amplicon sizes, the ratios of the copy numbers of the two primer pairs (SSIIb 3/4 and Le1n02/03) decreased with time in a heat-treated processing model using an autoclave. In this report, we suggest that the degradation level of DNA in processed foods could be estimated from these ratios, and the probability of GM quantification could be experimentally predicted from the results of the trial producing.     

DNA-based identification of spices: DNA isolation, whole genome amplification, and polymerase chain reaction

Usually spices are identified morphologically using simple methods like magnifying glasses or microscopic instruments. On the other hand, molecular biological methods like the polymerase chain reaction (PCR) enable an accurate and specific detection also in complex matrices. Generally, the origins of spices are plants with diverse genetic backgrounds and relationships. The processing methods used for the production of spices are complex and individual. Consequently, the development of a reliable DNA-based method for spice analysis is a challenging intention. However, once established, this method will be easily adapted to less difficult food matrices. In the current study, several alternative methods for the isolation of DNA from spices have been developed and evaluated in detail with regard to (i) its purity (photometric), (ii) yield (fluorimetric methods), and (iii) its amplifiability (PCR). Whole genome amplification methods were used to preamplify isolates to improve the ratio between amplifiable DNA and inhibiting substances. Specific primer sets were designed, and the PCR conditions were optimized to detect 18 spices selectively. Assays of self-made spice mixtures were performed to proof the applicability of the developed methods.     

Application of a real time polymerase chain reaction method to detect castor toxin contamination in fluid milk and eggs

The castor seed contains ricin, which is one of the most potent biological toxins and is widely considered to be a threat agent for bioterrorism. In this study, a rapid and sensitive PCR method was applied to the detection of castor contamination in milk and liquid egg samples. The targeting gene sequence of the primer set, Ricin-F4/R4, was not found in either the bovine or chicken genome. Primers against a highly conserved sequence from the 18S ribosomal RNA gene were used as a positive control for DNA extraction and PCR reaction efficiency. The quantity and quality of DNA prepared from castor spiked or nonspiked milk and egg samples obtained from three different DNA extraction methods were compared. The cetyl trimethylammonium bromide (CTAB) method yielded the highest quality of DNA and is most suitable for the sensitive detection of castor DNA by real-time PCR in both milk and liquid egg matrixes. However, taking time and cost into consideration, a commercial kit designed for extraction of DNA from stool samples could be used as an alternative method for the routine extraction of DNA from milk for real-time PCR assays. The egg matrix was found to inhibit PCR amplification and interfere with two of the three methods tested for DNA extraction. Egg yolk had a greater negative effect on PCR amplification than the egg white matrix. Our results affirm the necessity of performing individual validations for each food matrix. Both real-time PCR systems used in this study, TaqMan and SYBR Green I dye, were capable of detecting 100 ng of castor acetone powder, corresponding to 5 ng of ricin, in 1 mL of milk or liquid egg, well below the toxic dose for humans. On the basis of these results, the real-time PCR method for detection of intentional castor contamination is applicable to milk and egg matrixes.     

A sensitive sandwich ELISA for the detection of trace amounts of cashew (Anacardium occidentale L.) nut in foods

Trace amounts of cashew nut protein can provoke severe allergic reactions in sensitive patients. Consequently, commercial food processors and regulatory agencies must be vigilant to prevent cashew nut cross-contamination among foods and ensure proper labeling. Toward this end, we have developed a sandwich enzyme-linked immunosorbent (ELISA) to detect the predominant cashew protein fraction (anacardein or cashew major protein, CMP) that can be extracted in aqueous buffer from food matrixes. Protein G-purified goat antiwhole cashew extract IgG and rabbit anti-CMP IgG were used as capture and secondary antibodies, respectively. Immunoadsorption against several nut and seed proteins significantly minimized the inherent cross-reactivity of these reagents. Food samples spiked with cashew flour and CMP were extracted and tested in a sandwich ELISA where standard curves were based on reactivity with CMP. The assay was optimized to detect as little as 20 ng/mL (0.02 ppm) of CMP and was successfully used to quantify CMP, and thus cashew, in various food matrixes.     

小麦AB基因组中一个重复序列的分离、定位与应用

利用102对微卫星引物对5份黑麦（Secale）、4份普通小麦（Triticum aestivum）和1份分枝小黑麦（Triticale）进行SSR分析,引物Xgwm614能在分枝小黑麦中扩增出一个387bp的特异DNA片段（记为FZ387,GenBank登录号为EF179137）,而黑麦未能扩增出。序列比对结果显示该片段与一粒小麦（T. monococcum）（AY485644）和栽培二粒小麦（T. turgidum）（AY494981）A基因组中Gypsy Ty3-LTR反转座子fatima的一部分分别有94%和95%同源性。根据序列同源性比对结果,在FZ387内部设计1对特异引物FaF和FaR。引物Xgwm614F和FaR能在含有A基因组的物种中扩增出约350bp的条带（记为A350）,而其不含A基因组的物种都未扩增出该条带。利用小麦二体和端体代换系材料对其进行定位,结果显示该片段分布在所有A染色体的长臂和断臂上。此外,引物FaF和Xgwm614R能在含有A、B或AB基因组的物种中扩增出约350bp的条带（记为AB350）,而不含AB基因组的材料未扩增出目标条带。利用这两对特异引物对小麦属近缘物种进行PCR扩增,发现只有中国春能够扩增出A350和AB350。序列比对结果和FZ387两侧SSR引物结合区的规律性变化表明该反转座子在进化上可能存在属间多样性和属内相似性。A350和AB350也可以分别作为分子标记检测A染色体和AB染色体。     

Development of a seven-target multiplex PCR for the simultaneous detection of transgenic soybean and maize in feeds and foods

The detection of genetically modified organisms (GMOs) in food and feed is an important issue for all the subjects involved in raw material control, food industry, and distribution. Because the number of GMOs authorized in the EU increased during the past few years, there is a need for methods that allow a rapid screening of products. In this paper, we propose a method for the simultaneous detection of four transgenic maize (MON810, Bt11, Bt 176, and GA21) and one transgenic soybean (Roundup Ready), which allows routine control analyses to be sped up. DNA was extracted either from maize and soybean seeds and leaves or reference materials, and the recombinant DNA target sequences were detected with 7 primer pairs, accurately designed to be highly specific for each investigated transgene. Cross and negative controls were performed to ensure the specificity of each primer pair. The method was validated on an interlaboratory ring test and good analytical parameters were obtained (LOD = 0.25%, Repeatability, (r) = 1; Reproducibility, (R) = 0.9). The method was then applied to a model biscuit made of transgenic materials baked for the purpose and to real samples such as feed and foodstuffs. On account of the high recognition specificity and the good detection limits, this multiplex PCR represents a fast and reliable screening method directly applicable in all the laboratories involved in raw material and food control.     

苹果基因邻近未知序列的PCR扩增与测序方法

本文提出的方法是首先利用已知基因序列设计三个巢式PCR引物p14、p15和p16,然后设计3’端为常见酶切位点、5’端为随机引物序列p17的9个酶切位点引物,用p14外引物与9个酶切位点引物进行第一轮PCR扩增,其中前5个反应的退火温度较低可以将酶切位点引物的5’端序列引入到PCR产物中,其余反应则以此PCR产物为模板采用较高的正常退火温度进行,退火温度较高是为了使整个酶切位点引物能稳定地结合到最初反应形成的产物上。为提高PCR产物的特异性和产量,用p15和p16引物分别与p17引物配对进行第二轮和第三轮PCR扩增,电泳检查发现PCR产物条带后进行纯化和测序。为保证序列的正确性,利用测得的DNA序列再设计新引物f3x与p14(或p15)配对,直接用提取的DNA为模板进行PCR扩增和测序。利用此方法成功地获得了苹果FPPS基因邻近的启动子序列528bp和5’UTR序列142bp,并已在GenBank注册（登录号FJ263960）。利用Blastn发现这是GenBank中首次发表苹果FPPS基因启动子序列。这说明用该方法获取基因邻近的未知序列是可行的。     

Qualitative and quantitative polymerase chain reaction analysis for genetically modified maize MON863

Qualitative and quantitative analytical methods were developed for the new event of genetically modified (GM) maize, MON863. One specific primer pair was designed for the qualitative polymerase chain reaction (PCR) method. The specificity and sensitivity of the designed primers were confirmed. PCR was performed on genomic DNAs extracted from MON863, other GM events, and cereal crops. Single PCR product was obtained from MON863 by the designed primer pair. Eight test samples including GM maize MON863 were prepared at 0.01 approximately 10% levels and analyzed by PCR. Limit of detection of the method was 0.01% for GM maize MON863. On the other hand, another specific primer pair and probe were also designed for quantitative method using a real-time polymerase chain reaction. As a reference molecule, a plasmid was constructed from a taxon-specific DNA sequence for maize, a universal sequence for a cauliflower mosaic virus (CaMV) 35S promoter used in most genetically modified organisms, and a construct-specific DNA sequence for the MON863 event. Six test samples of 0.1, 0.5, 1.0, 3.0, 5.0 and 10.0% of GM maize MON863 were quantitated for the validation of this method. At the 3.0% level, the bias (mean vs true value) for MON863 was 3.0%, and its relative standard deviation was 5.5%. Limit of quantitation of the method was 0.5%. These results show that the developed PCR methods can be used to qualitatively and quantitatively detect GM maize MON863.     

大鲵致病性嗜水气单胞菌SYBR GreenⅠ荧光定量PCR诊断试剂盒的研制

根据GenBank中致病性嗜水气单胞菌特异性的气溶素基因序列,设计1对引物,利用普通PCR技术扩增获得hlyA基因片段,并克隆到pMD-18T载体上作为阳性标准品。通过对SYBR GreenⅠ荧光定量PCR反应条件的优化,建立了快速检测致病性嗜水气单胞菌的SYBR GreenⅠ荧光定量诊断方法,以此为基础研制出试剂盒。试剂盒扩增产物的熔解曲线分析只出现1个单特异峰,无引物二聚体,对非致病性嗜水气单胞菌、弗氏柠檬酸杆菌、迟缓爱德华菌、柱状黄杆菌均无阳性信号扩增,重复性好,灵敏度可达1.0x101拷贝/uL。结果表明研制的致病性嗜水气单胞菌SYBR GreenⅠ实时荧光定量PCR试剂盒具有特异、灵敏、快速、重复性好等特点,适合于大鲵临床样品的检测。     

Comparison of DNA extraction methods and development of duplex PCR and real-time PCR to detect tomato, carrot, and celery in food

Traceability is of particular importance for those persons who suffer allergy or intolerance to some food component(s) and need a strict avoidance of the allergenic food. In this paper, methodologies are described to fingerprint the presence of allergenic species such as carrot, tomato, and celery by DNA detection. Three DNA extraction methods were applied on vegetables and foods containing or not containing the allergens, and the results were compared and discussed. Fast SYBR Green DNA melting curve temperature analyses and duplex PCR assays with internal control have been developed for detection of these allergenic vegetables and have been tested on commercial foods. Spiking food experiments were also performed, assessing that limits of detection (LOD) of 1 mg/kg for carrot and tomato DNA and 10 mg/kg for celery DNA have been reached.     

Identification of gadoid species in fish meat by polymerase chain reaction (PCR) on genomic DNA

Identification of fish species is significant due to the increasing interest of consumers in the meat of sea fish. Methods focusing on fish species identification help to reveal fraudulent substitution among economically important gadoid species in commercial seafood products. The objective of this work was to develop a conventional PCR method for the differentiation of the following gadoid fish species in fish products: Alaska pollack ( Theragra chalcogramma), blue whiting ( Micromesistius poutassou), hake spp. ( Merluccius spp.), Atlantic cod ( Gadus morhua), saithe ( Pollachius virens), and whiting ( Merlangius merlangus). The species-specific primer pairs for gadoid species determination were based on the partial pantophysin I ( PanI) genomic sequence. Sequence identification was confirmed by cloning and sequencing of the PCR products obtained from the species considered. For the simultaneous detection of Alaska pollack, blue whiting, and hake spp., a quadruplex PCR system was constructed. Other gadoid species were detected in separate PCR reactions. After optimization of the reactions, the developed PCR systems were used for the analysis of codfish samples obtained from the Czech market and the customs' laboratories. This method represents an alternative approach in the use of genomic DNA for the identification of fish species. This method is rapid, simple, and reliable without the need for further confirmative methods. Furthermore, the identification of a mixture of more than one species is possible. The PCR system has been optimized for routine diagnostic purposes.     

Real-time polymerase chain reaction quantification of the transgenes for roundup ready corn and roundup ready soybean in soil samples

A method for quantification of recombinant DNA for Roundup Ready (RR) corn and RR soybean in soil samples is described. Soil DNA from experimental field samples was extracted using a soil DNA extraction kit with a modified protocol. For the detection and quantification of recombinant DNA of RR corn and RR soybean, a molecular beacon and two pairs of specific primers were designed to differentially target recombinant DNA in these two genetically modified crops. Soil DNA extracts were spiked with RR corn or RR soybean DNA, and recombinant DNA was quantified using real-time PCR with a molecular beacon. As few as one copy of RR corn genome or one copy of RR soybean genome was detected in the soil DNA extract.     

Earthworm primers for DNA-based gut content analysis and their cross-reactivity in a multi-species system

Specific primers can be used in polymerase chain reactions (PCR) to amplify prey DNA from the gut content of generalist predators at high specificity and sensitivity. A prerequisite for applying this approach to field studies, however, is to confirm that primers are actually targeting specific prey species or prey groups and do not produce false positive results by amplifying DNA either from predator species or from the wide range of potential alternative prey found under natural conditions. Here, we report on a new group-specific primer pair for earthworms designed from cytochrome oxidase c subunit 1 (COI) sequences of 11 earthworm species found in Central Europe that can be used to detect consumption of earthworms by invertebrate predators. Besides inter-specific also considerable intra-specific variation was found for COI sequences among most of the earthworm species. We, therefore, combined a universal forward primer with an earthworm-specific reverse primer which amplified a 523 bp product from all 11 species tested. Earthworm DNA amplification was also successful in the presence of excess DNA of a predator species. The primer pair was tested against 82 non-target invertebrate species commonly found in the same habitats, including potential prey for generalist predators and predators themselves. The earthworm primer was highly specific: only one of the non-target species showed a product of similar length as the earthworms, whereas PCR with 12 non-target species produced amplicons whose length differed from that of earthworms. We conclude that the new primer will be a useful tool to investigate the role earthworms play as a food resource in soil food-webs. Moreover, we suggest that future studies utilizing DNA-based approaches for prey detection should select non-target species for cross-reactivity tests according to their abundance and importance rather than choosing representatives of taxonomic units; this will help validate the results achieved using species- or group-specific primers and guarantee their meaningful ecological interpretation.