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1.
Real-time polymerase chain reaction is currently being used for the identification and quantification of plant and animal species as well as microorganisms in food or feed samples based on the amplification of specific sequences of low copy genes. We report here the development of a new real-time PCR method for the detection and quantification of the pea (Pisum sativum) based on the amplification of a specific region of the legS gene. The specificity was evaluated in a wide range of plant species (51 varieties of Pisum sp., and 32 other plant species and varieties taxonomically related or nonrelated). The method allows the detection and quantification of as low as 21.6 pg of DNA, which corresponds to 5 haploid genome copies. The system has been shown to be sensitive, reproducible and 100% specific for the rapid detection and quantification of pea DNA in processed food and feed samples, being therefore suitable for high-throughput analysis.  相似文献   

2.
We report the development of a duplex real-time Polymerase Chain Reaction (PCR) for the simultaneous detection and quantification of wheat- and barley-derived DNA. We used a single primer pair to amplify the single-copy gene PKABA1 from wheat and barley, using minor-groove-binding probes to distinguish between the two cereals. The assay was fully specific, and different wheat and barley cultivars exhibited similar Ct values, indicating stability across cultivars with respect to allelic and copy number composition. The limits of detection were 5 and 10 PCR-forming units for wheat and barley, respectively, making the duplex assay as sensitive as other singleplex reference gene systems published. We were able to detect both wheat and barley simultaneously in real food samples, and the duplex assay is considered to be suitable as an endogenous reference gene system for the detection and quantification of wheat and barley in genetically modified organisms (GMO) and other food and feed analyses.  相似文献   

3.
Compliance with the European allergen labeling legislation (Directive 2007/68/EC) is only possible when coupled with appropriate methods to detect allergens in food. The aim of the current study was to develop new real-time PCR assays for the detection of hazelnut and soy and evaluate these assays via comparison with commercially available kits. Although the new assays were not as sensitive as the commercial qualitative assays, they proved to be more specific. Moreover, the cross-reactivity study indicated contamination of some of the food products used with either hazelnut or soy, which presents a risk for the allergic consumer. The assays were able to quantify as few as 5-15 genome copies. This unit, used to express analytical results for allergen detection by means of PCR, needs to be converted to a unit expressing the amount of allergenic ingredient in order to be informative. This study emphasizes that the use of real-time PCR for allergen quantification is complicated by the lack of appropriate reference materials for allergens.  相似文献   

4.
Polymerase chain reaction (PCR) methods are very useful techniques for the detection and quantification of genetically modified organisms (GMOs) in food samples. These methods rely on the amplification of transgenic sequences and quantification of the transgenic DNA by comparison to an amplified reference gene. Reported here is the development of specific primers for the rapeseed (Brassica napus) BnACCg8 gene and PCR cycling conditions suitable for the use of this sequence as an endogenous reference gene in both qualitative and quantitative PCR assays. Both methods were assayed with 20 different rapeseed varieties, and identical amplification products were obtained with all of them. No amplification products were observed when DNA samples from other Brassica species, Arabidopsis thaliana, maize, and soybean were used as templates, which demonstrates that this system is specific for rapeseed. In real-time quantitative PCR analysis, the detection limit was as low as 1.25 pg of DNA, which indicates that this method is suitable for use in processed food samples which contain very low copies of target DNA.  相似文献   

5.
Polymerase chain reaction (PCR) methods have been the main technical support for the detection of genetically modified organisms (GMOs). To date, GMO-specific PCR detection strategies have been developed basically at four different levels, such as screening-, gene-, construct-, and event-specific detection methods. Event-specific PCR detection method is the primary trend in GMO detection because of its high specificity based on the flanking sequence of exogenous integrant. GM canola, event T45, with tolerance to glufosinate ammonium is one of the commercial genetically modified (GM) canola events approved in China. In this study, the 5'-integration junction sequence between host plant DNA and the integrated gene construct of T45 canola was cloned and revealed by means of TAIL-PCR. Specific PCR primers and TaqMan probes were designed based upon the revealed sequence, and qualitative and quantitative TaqMan real-time PCR detection assays employing these primers and probe were developed. In qualitative PCR, the limit of detection (LOD) was 0.1% for T45 canola in 100 ng of genomic DNA. The quantitative PCR assay showed limits of detection and quantification (LOD and LOQ) of 5 and 50 haploid genome copies, respectively. In addition, three mixed canola samples with known GM contents were detected employing the developed real-time PCR assay, and expected results were obtained. These results indicated that the developed event-specific PCR methods can be used for identification and quantification of T45 canola and its derivates.  相似文献   

6.
The genetically modified (GM) food/feed quantification depends on the reliable detection systems of endogenous reference genes. Currently, four endogenous reference genes including sucrose phosphate synthase (SPS), GOS9, phospholipase D (PLD), and ppi phosphofructokinase (ppi-PPF) of rice have been used in GM rice detection. To compare the applicability of these four rice reference genes in quantitative PCR systems, we analyzed the target nucleotide sequence variation in 58 conventional rice varieties from various geographic and phylogenic origins, also their quantification performances were evaluated using quantitative real-time PCR and GeNorm analysis via a series of statistical calculation to get a "M value" which is negative correlation with the stability of genes. The sequencing analysis results showed that the reported GOS9 and PLD taqman probe regions had detectable single nucleotide polymorphisms (SNPs) among the tested rice cultivars, while no SNPs were observed for SPS and ppi-PPF amplicons. Also, poor quantitative performance was detectable in these cultivars with SNPs using GOS9 and PLD quantitative PCR systems. Even though the PCR efficiency of ppi-PPF system was slightly lower, the SPS and ppi-PPF quantitative PCR systems were shown to be applicable for rice endogenous reference assay with less variation among the C(t) values, good reproducibility in quantitative assays, and the low M values by the comprehensive quantitative PCR comparison and GeNorm analysis.  相似文献   

7.
With the development of genetically modified organism (GMO) detection techniques, the Polymerase Chain Reaction (PCR) technique has been the mainstay for GMO detection, and real-time PCR is the most effective and important method for GMO quantification. An event-specific detection strategy based on the unique and specific integration junction sequences between the host plant genome DNA and the integrated gene is being developed for its high specificity. This study establishes the event-specific detection methods for TC1507 and CBH351 maizes. In addition, the event-specific TaqMan real-time PCR detection methods for another seven GM maize events (Bt11, Bt176, GA21, MON810, MON863, NK603, and T25) were systematically optimized and developed. In these PCR assays, the fluorescent quencher, TAMRA, was dyed on the T-base of the probe at the internal position to improve the intensity of the fluorescent signal. To overcome the difficulties in obtaining the certified reference materials of these GM maizes, one novel standard reference molecule containing all nine specific integration junction sequences of these GM maizes and the maize endogenous reference gene, zSSIIb, was constructed and used for quantitative analysis. The limits of detection of these methods were 20 copies for these different GM maizes, the limits of quantitation were about 20 copies, and the dynamic ranges for quantification were from 0.05 to 100% in 100 ng of DNA template. Furthermore, nine groups of the mixed maize samples of these nine GM maize events were quantitatively analyzed to evaluate the accuracy and precision. The accuracy expressed as bias varied from 0.67 to 28.00% for the nine tested groups of GM maize samples, and the precision expressed as relative standard deviations was from 0.83 to 26.20%. All of these indicated that the established event-specific real-time PCR detection systems and the reference molecule in this study are suitable for the identification and quantification of these GM maizes.  相似文献   

8.
The most widely accepted methods for accurate quantitative detection of genetically modified organisms rely on real-time PCR. Various detection chemistries are available for real-time PCR. They include sequence-unspecific DNA labeling dyes such SYBR-Green I and the use of both universal (e.g., AmpliFluor) and sequence-specific double-labeled probes, the latter comprising hybridization (e.g., Molecular Beacon) and hydrolysis (e.g., TaqMan or MGB) probes. Also, new real-time PCR devices and reagents allowing fast cycling reactions exist. Five Mon810 real-time PCR assays were developed in which the event specificity was based on the detection of transgene and plant rearranged sequences found to 3' flank the insertion site. Every assay was specifically designed for one particular detection chemistry, that is, AmpliFluor, Molecular Beacon, MGB, TaqMan, and SYBR-Green I. When possible, the assays were adapted to fast cycling mode. All assays displayed satisfactory performance parameters, although Molecular Beacon, MGB, and TaqMan chemistries were the most suitable for quantification purposes in both conventional and fast cycling modes.  相似文献   

9.
We report the development of an oligonucleotide microarray for the simultaneous detection of six important cereal food plant species from the Poaceae based on the chloroplast trnL intron sequence. We used universal primers to amplify the trnL intron from wheat, rye, barley, oat, rice, and maize, followed by a cyclic labeling of oligonucleotides probes and subsequent hybridization to an oligonucleotide microarray. In single taxon analyses, positive signals were produced with a high signal-to-noise ratio. The assay also enabled the analysis of mixed samples. The results obtained for real food samples were in agreement with the ingredient labels, but positive results for grains not declared on the ingredients list were observed in three out of 10 samples, which indicates that the final products and/or the declared ingredients were probably botanically impure or contaminated. The combination of the sensitivity of a universal polymerase chain reaction with the specificity of the labeling reaction allows this protocol to be applied in routine analyses of food samples, as demonstrated by successful analysis of processed composite food products.  相似文献   

10.
Sensitive and accurate testing for trace amounts of biotechnology-derived DNA from plant material is the prerequisite for detection of 1% or 0.5% genetically modified ingredients in food products or raw materials thereof. Compared to ELISA detection of expressed proteins, real-time PCR (RT-PCR) amplification has easier sample preparation and detection limits are lower. Of the different methods of DNA preparation CTAB method with high flexibility in starting material and generation of sufficient DNA with relevant quality was chosen. Previous RT-PCR data generated with the SYBR green detection method showed that the method is highly sensitive to sample matrices and genomic DNA content influencing the interpretation of results. Therefore, this paper describes a real-time DNA quantification based on the TaqMan probe method, indicating high accuracy and sensitivity with detection limits of lower than 18 copies per sample applicable and comparable to highly purified plasmid standards as well as complex matrices of genomic DNA samples. The results were evaluated with ValiData for homology of variance, linearity, accuracy of the standard curve, and standard deviation.  相似文献   

11.
For implementation of the issued regulations and labeling policies for genetically modified organism (GMO) supervision, the polymerase chain reaction (PCR) method has been widely used due to its high specificity and sensitivity. In particular, use of the event-specific PCR method based on the flanking sequence of transgenes has become the primary trend. In this study, both qualitative and quantitative PCR methods were established on the basis of the 5' flanking sequence of transgenic soybean A2704-12 and the 3' flanking sequence of transgenic soybean A5547-127, respectively. In qualitative PCR assays, the limits of detection (LODs) were 10 copies of haploid soybean genomic DNA for both A2704-12 and A5547-127. In quantitative real-time PCR assays, the LODs were 5 copies of haploid soybean genomic DNA for both A2704-12 and A5547-127, and the limits of quantification (LOQs) were 10 copies for both. Low bias and acceptable SD and RSD values were also achieved in quantification of four blind samples using the developed real-time PCR assays. In addition, the developed PCR assays for the two transgenic soybean events were used for routine analysis of soybean samples imported to Shanghai in a 6 month period from October 2010 to March 2011. A total of 27 lots of soybean from the United States and Argentina were analyzed: 8 lots from the Unites States were found to have the GM soybean A2704-12 event, and the GM contents were <1.5% in all eight analyzed lots. On the contrary, no GM soybean A5547-127 content was found in any of the eight lots. These results demonstrated that the established event-specific qualitative and quantitative PCR methods could be used effectively in routine identification and quantification of GM soybeans A2704-12 and A5547-127 and their derived products.  相似文献   

12.
Qualitative and quantitative Polymerase Chain Reaction (PCR) systems aimed at the specific detection and quantification of common wheat DNA are described. Many countries have issued regulations to label foods that include genetically modified organisms (GMOs). PCR technology is widely recognized as a reliable and useful technique for the qualitative and quantitative detection of GMOs. Detection methods are needed to amplify a target GM gene, and the amplified results should be compared with those of the corresponding taxon-specific reference gene to obtain reliable results. This paper describes the development of a specific DNA sequence in the waxy-D1 gene for common wheat (Triticum aestivum L.) and the design of a specific primer pair and TaqMan probe on the waxy-D1 gene for PCR analysis. The primers amplified a product (Wx012) of 102 bp. It is indicated that the Wx012 DNA sequence is specific to common wheat, showing homogeneity in qualitative PCR results and very similar quantification accuracy along 19 distantly related common wheat varieties. In Southern blot and real-time PCR analyses, this sequence showed either a single or a low number of copy genes. In addition, by qualitative and quantitative PCR using wx012 primers and a wx012-T probe, the limits of detection of the common wheat genome were found to be about 15 copies, and the reproducibility was reliable. In consequence, the PCR system using wx012 primers and wx012-T probe is considered to be suitable for use as a common wheat-specific taxon-specific reference gene in DNA analyses, including GMO tests.  相似文献   

13.
Toward the development of reliable qualitative and quantitative Polymerase Chain Reaction (PCR) detection methods of transgenic tomatoes, one tomato (Lycopersicon esculentum) species specific gene, LAT52, was selected and validated as suitable for using as an endogenous reference gene in transgenic tomato PCR detection. Both qualitative and quantitative PCR methods were assayed with 16 different tomato varieties, and identical amplified products or fluorescent signals were obtained with all of them. No amplified products and fluorescent signals were observed when DNA samples from 20 different plants such as soybean, maize, rapeseed, rice, and Arabidopsis thaliana were used as templates. These results demonstrated that the amplified LAT52 DNA sequence was specific for tomato. Furthermore, results of Southern blot showed that the LAT52 gene was a single-copy gene in the different tested tomato cultivars. In qualitative and quantitative PCR analysis, the detection sensitivities were 0.05 and 0.005 ng of tomato genomic DNA, respectively. In addition, two real-time assays employing this gene as an endogenous reference gene were established, one for the quantification of processed food samples derived from nontransgenic tomatoes that contained degraded target DNA and the other for the quantification of the junction region of CaMV35s promoter and the anti-sense ethylene-forming enzyme (EFE) gene in transgenic tomato Huafan No. 1 samples. All of these results indicated that the LAT52 gene could be successfully used as a tomato endogenous reference gene in practical qualitative and quantitative detection of transgenic tomatoes, even for some processed foods derived from transgenic and nontransgenic tomatoes.  相似文献   

14.
With the development of transgenic crops, many countries have issued regulations to label the genetically modified organisms (GMOs) and their derived products. Polymerase Chain Reaction (PCR) methods are thought to be reliable and useful techniques for qualitative and quantitative detection of GMOs. These methods generally need to amplify the transgene and compare the amplified result with that of the corresponding reference gene to obtain reliable results. In this article, we reported the development of specific primers and probe for the rice (Oryza sativa) sucrose phosphate synthase (SPS) gene and PCR cycling conditions suitable for the use of this sequence as an endogenous reference gene in both qualitative and quantitative PCR assays. Both methods were assayed with 13 different rice varieties, and identical amplification products were obtained with all of them. No amplification products were observed when DNA samples from other species, such as wheat, maize, barley, tobacco, soybean, rapeseed, tomato, sunflower, carrot, pepper, eggplant, lupine, mung bean, plum, and Arabidopsis thaliana, were used as templates, which demonstrated that this system was specific for rice. In addition, the results of the Southern blot analysis confirmed that the SPS gene was a single copy in the tested rice varieties. In qualitative and quantitative PCR analyses, the detection sensitivities were 0.05 and 0.005 ng of rice genomic DNA, respectively. To test the practical use of this SPS gene as an endogenous reference gene, we have also quantified the beta-glucuronidase (GUS) gene in transgenic rice using this reference gene. These results indicated that the SPS gene was species specific, had one copy number, and had a low heterogeneity among the tested cultivars. Therefore, this gene could be used as an endogenous reference gene of rice and the optimized PCR systems could be used for practical qualitative and quantitative detection of transgenic rice.  相似文献   

15.
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.  相似文献   

16.
As more genetically modified plant events are approved for commercialization worldwide, the event-specific PCR method has become the key method for genetically modified organism (GMO) identification and quantification. This study reveals the 3' flanking sequence of the exogenous integration of Oxy-235 canola employing thermal asymmetric interlaced PCR (TAIL-PCR). On the basis of the revealed 3' flanking sequence, PCR primers and TaqMan probe were designed and qualitative and quantitative PCR assays were established for Oxy-235 canola. The specificity and limits of detection (LOD) and quantification (LOQ) of these two PCR assays were validated to as low as 0.1% for the relative LOD of qualitative PCR assay; the absolute LOD and LOQ were low to 10 and 20 copies of canola genomic DNA in quantitative PCR assay, respectively. Furthermore, ideal quantified results were obtained in the practical canola sample detection. All of the results indicate that the developed qualitative and quantitative PCR methods based on the revealed 3' integration flanking sequence are suitable for GM canola Oxy-235 identification and quantification.  相似文献   

17.
Because of the genetically modified organisms (GMOs) labeling policies issued in many countries and areas, polymerase chain reaction (PCR) methods were developed for the execution of GMO labeling policies, such as screening, gene specific, construct specific, and event specific PCR detection methods, which have become a mainstay of GMOs detection. The event specific PCR detection method is the primary trend in GMOs detection because of its high specificity based on the flanking sequence of the exogenous integrant. This genetically modified maize, MON863, contains a Cry3Bb1 coding sequence that produces a protein with enhanced insecticidal activity against the coleopteran pest, corn rootworm. In this study, the 5'-integration junction sequence between the host plant DNA and the integrated gene construct of the genetically modified maize MON863 was revealed by means of thermal asymmetric interlaced-PCR, and the specific PCR primers and TaqMan probe were designed based upon the revealed 5'-integration junction sequence; the conventional qualitative PCR and quantitative TaqMan real-time PCR detection methods employing these primers and probes were successfully developed. In conventional qualitative PCR assay, the limit of detection (LOD) was 0.1% for MON863 in 100 ng of maize genomic DNA for one reaction. In the quantitative TaqMan real-time PCR assay, the LOD and the limit of quantification were eight and 80 haploid genome copies, respectively. In addition, three mixed maize samples with known MON863 contents were detected using the established real-time PCR systems, and the ideal results indicated that the established event specific real-time PCR detection systems were reliable, sensitive, and accurate.  相似文献   

18.
Chemical composition and in vitro digestion properties of select whole grains, before and after processing, and their components were measured. Substrates included barley, corn, oat, rice, and wheat. In addition to whole grain flours, processed substrates also were tested as were corn bran, oat bran, wheat bran, and wheat germ. Processing of most substrates resulted in higher dry matter and digestible starch and lower resistant starch concentrations. Dietary fiber fractions varied among substrates with processing. Digestion profiles for most substrates correlated well with their chemical composition. Corn bran and rice substrates were the least fermentable. Extrusion rendered barley, corn, and wheat more hydrolytically digestible and barley and oat more fermentatively digestible. Except for corn bran, all components had greater or equal fermentability compared with their native whole grains. Understanding digestion characteristics of whole grains and their components will allow for more accurate utilization of these ingredients in food systems.  相似文献   

19.
We have tested and compared the performance of 12 different assays representing four different real-time polymerase chain reaction (PCR) chemistries in the context of genetically modified organism detection. Several different molecular beacon, SYBR Green, TaqMan, and MGB assays were designed for the event specific detection and quantification of the 3' integration junction of GTS 40-3-2 (Roundup Ready) soybean. Sensitivity as well as robustness in the presence of background DNA were tested. None of the PCR-based approaches appeared to be significantly better than any of the other, but the molecular beacon assays had the lowest efficiency and also seemed more sensitive to changes in experimental setup.  相似文献   

20.
Anthocyanin pigments from a wide variety of edible and ornamental black, blue, pink, purple, red, and white wheat, barley, corn, rice, and wild rice were identified and quantified to evaluate their potential as natural colorants or functional food ingredients. The total anthocyanin contents varied significantly and exhibited a range of 7-3276 microg/g. Some grains, such as red rice and black rice, contained a limited number of pigments, whereas others, such as blue, pink, purple, and red corns, had complex anthocyanin profiles. Of the 42 anthocyanin compounds observed, 9 were characterized by comparison of the spectroscopic and chromatographic properties with those of authentic standards. The remaining compounds were tentatively identified on the basis of spectroscopic properties and electrospray ionization mass spectra. The most abundant anthocyanins were cyanidin 3-glucoside in black and red rices and in blue, purple, and red corns, pelargonidin 3-glucoside in pink corn, and delphinidin 3-glucoside in blue wheat.  相似文献   

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