首页 | 本学科首页   官方微博 | 高级检索  
相似文献
 共查询到20条相似文献，搜索用时 31 毫秒
1.
Quality assurance is a major issue in the food industry. The authenticity of food ingredients and their traceability are required by consumers and authorities. Plant species such as barley (Hordeum vulgare), rice (Oryza sativa), sunflower (Helianthus annuus), and wheat (Triticum aestivum) are very common among the ingredients of many processed food products; therefore the development of specific assays for their specific detection and quantification are needed. Furthermore, the production and trade of genetically modified lines from an increasing number of plant species brings about the need for control within research, environmental risk assessment, labeling/legal, and consumers' information purposes. We report here the development of four independent real-time polymerase chain reaction (PCR) assays suitable for identification and quantification of four plant species (barley, rice, sunflower, and wheat). These assays target gamma-hordein, gos9, helianthinin, and acetyl-CoA carboxylase sequences, respectively, and were able to specifically detect and quantify DNA from the target plant species. In addition, the simultaneous amplification of RALyase allowed bread from durum wheat to be distinguished. Limits of detection were 1 genome copy for barley, sunflower, and wheat and 3.3 copies for rice real-time PCR systems, whereas limits of quantification were 10 genome copies for barley, sunflower, or wheat and approximately 100 haploid genomes for rice real-time PCR systems. Real-time PCR cycling conditions of the four assays were stated as standard to facilitate their use in routine laboratory analyses. The assays were finally adapted to conventional PCR for detection purposes, with the exception of the wheat assay, which detects rye simultaneously with similar sensitivity in an agarose gel.  相似文献   

2.
The labeling of products containing genetically modified organisms (GMO) is linked to their quantification since a threshold for the presence of fortuitous GMOs in food has been established. This threshold is calculated from a combination of two absolute quantification values: one for the specific GMO target and the second for an endogenous reference gene specific to the taxon. Thus, the development of reliable methods to quantify GMOs using endogenous reference genes in complex matrixes such as food and feed is needed. Plant identification can be difficult in the case of closely related taxa, which moreover are subject to introgression events. Based on the homology of beta-fructosidase sequences obtained from public databases, two couples of consensus primers were designed for the detection, quantification, and differentiation of four Solanaceae: potato (Solanum tuberosum), tomato (Solanum lycopersicum), pepper (Capsicum annuum), and eggplant (Solanum melongena). Sequence variability was studied first using lines and cultivars (intraspecies sequence variability), then using taxa involved in gene introgressions, and finally, using taxonomically close taxa (interspecies sequence variability). This study allowed us to design four highly specific TaqMan-MGB probes. A duplex real time PCR assay was developed for simultaneous quantification of tomato and potato. For eggplant and pepper, only simplex real time PCR tests were developed. The results demonstrated the high specificity and sensitivity of the assays. We therefore conclude that beta-fructosidase can be used as an endogenous reference gene for GMO analysis.  相似文献   

3.
Partial sequencing of the γ-gliadin gene of 62 spelt and 14 soft wheat cultivars was performed. Fifty-six of the 62 spelt cultivars and 13 of the 14 soft wheat cultivars were shown to exhibit the typical spelt or soft wheat γ-gliadin sequence, respectively. Exceptions were ascribed to crossbreeding of soft wheat and spelt. Using the typical soft wheat γ-gliadin sequence, two alternative DNA-based analytical methods were developed for the detection and quantification of spelt flour "adulteration" with soft wheat. A simple and fast detection of soft wheat in spelt flours could be achieved by restriction fragment length (RFLP) analysis. In combination with lab-on-a-chip capillary gel electrophoresis (LOC-CE) the soft wheat proportion could be estimated. Heteroduplex formation served as additional confirmation for the presence of spelt besides soft wheat. Hence, RFLP-LOC-CE constitutes a perfect analysis tool for the quality control of cereal seeds and pure cultivars. A precise quantification of soft wheat "adulterations" in spelt flour down to 1% could be achieved by the developed real-time PCR method. The calibration parameters of the real-time PCR assay fulfilled the minimum performance requirements of the European Network of GMO (genetically modified organisms) Laboratories (ENGL).  相似文献   

4.
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   

5.
To enforce the labeling regulations of genetically modified organisms (GMOs), the application of reference molecules as calibrators is becoming essential for practical quantification of GMOs. However, the reported reference molecules with tandem marker multiple targets have been proved not suitable for duplex PCR analysis. In this study, we developed one unique plasmid molecule based on one pMD-18T vector with three exogenous target DNA fragments of Roundup Ready soybean GTS 40-3-2 (RRS), that is, CaMV35S, NOS, and RRS event fragments, plus one fragment of soybean endogenous Lectin gene. This Lectin gene fragment was separated from the three exogenous target DNA fragments of RRS by inserting one 2.6 kb DNA fragment with no relatedness to RRS detection targets in this resultant plasmid. Then, we proved that this design allows the quantification of RRS using the three duplex real-time PCR assays targeting CaMV35S, NOS, and RRS events employing this reference molecule as the calibrator. In these duplex PCR assays, the limits of detection (LOD) and quantification (LOQ) were 10 and 50 copies, respectively. For the quantitative analysis of practical RRS samples, the results of accuracy and precision were similar to those of simplex PCR assays, for instance, the quantitative results were at the 1% level, the mean bias of the simplex and duplex PCR were 4.0% and 4.6%, respectively, and the statistic analysis ( t-test) showed that the quantitative data from duplex and simplex PCR had no significant discrepancy for each soybean sample. Obviously, duplex PCR analysis has the advantages of saving the costs of PCR reaction and reducing the experimental errors in simplex PCR testing. The strategy reported in the present study will be helpful for the development of new reference molecules suitable for duplex PCR quantitative assays of GMOs.  相似文献   

6.
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   

7.
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   

8.
We identified volatile compounds of barley flour and determined the variation in volatile compound profiles among different types and varieties of barley. Volatile compounds of 12 barley and two wheat cultivars were analyzed using solid phase microextraction (SPME) and gas chromatography. Twenty-six volatiles comprising aldehydes, ketones, alcohols, and a furan were identified in barley. 1-Octen-3-ol, 3-methylbutanal, 2-methylbutanal, hexanal, 2-hexenal, 2-heptenal, 2-nonenal, and decanal were identified as key odorants in barley as their concentration exceeded their odor detection threshold in water. Hexanal (46-1269 microg/L) and 1-pentanol (798-1811 microg/L) were the major volatile compounds in barley cultivars. In wheat, 1-pentanol (723-748 microg/L) was a major volatile. Hulled barley had higher total volatile, aldehyde, ketone, alcohol, and furan contents than hulless barley, highlighting the importance of the husk in barley grain aroma. The proanthocyanidin-free varieties generally showed higher total volatile and aldehyde contents than wild-type varieties, potentially due to decreased antioxidant activity by the absence of proanthocyanidins.  相似文献   

9.
Genetically modified (GM) alfalfa (Medicago sativa) was marketed for the first time in 2005. For countries with established thresholds for GM plants, methods to detect and quantify their adventitious presence are required. We selected acetyl CoA carboxylase as a reference gene for the detection and quantification of GM alfalfa. Two qualitative polymerase chain reaction (PCR) assays (Acc1 and Acc2) were designed to detect alfalfa. Both were specific to alfalfa, amplifying DNA from 12 separate cultivars and showing negative results for PCR of 15 nonalfalfa plants. The limits of detection for Acc1 and Acc2 were 0.2 and 0.01%, respectively. A quantitative real-time PCR assay was also designed, having high linearity (r > 0.99) over alfalfa standard concentrations ranging from 100 to 2.0 x 10(5) pg of alfalfa DNA per PCR. The real-time PCR assay was effective in quantifying alfalfa DNA from forage- and concentrate-based mixed diets containing different amounts of alfalfa meal.  相似文献   

10.
  相似文献   

11.
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   

12.
The use of chlorate as a nitrate analogue to screen soft red winter wheat (Triticum aestivum L.) cultivars for differences in nitrate reductase activity (NRA) was studied by adding potassium chlorate to a hydroponic nutrient solution in which wheat seedlings were growing. After 14 days, leaf symptoms indicating chlorate‐induced toxicity were rated. It was hypothesized that wheat plants which were susceptible to chlorate‐induced toxicity reduced chlorate and nitrate more rapidly than did resistant plants. In experiments testing the potential of this assay, wheat and barley (Hordeum vulgare L.) cultivars previously reported to have low NRA were less susceptible to chlorate‐induced toxicity than were cultivars reported to have high NRA. The assay was used to screen 15 soft red winter wheat cultivars for differences in sensitivity to chlorate‐induced toxicity. Variable toxic reactions were observed both among and within the cultivars. To determine whether the within‐cultivar variation was environmental or genetic, single plant selections for contrasting chlorate response were made, and bulked progeny were rescreened. In eight of 15 cultivars, the contrasting selections were different for chlorate‐induced toxic response, indicating heterogeneity for this trait within these eight cultivars. These chlorate‐selected lines may also be near‐isogenic lines for NRA. Seedling screening of wheat for chlorate response may be useful for identification of high NRA breeding lines.  相似文献   

13.
The natural content of ochratoxin A in grain samples of 6 barley, 2 bread wheat and 1 durum wheat cultivars varied from <0.1 to 0.4 ng/g grain. Samples of the analysed cultivars were surface sterilized and kept in humidity chambers at 20°C and water activity (aw) 0.75 or aw 0.85 for 8 days. For both environments, the resulting grain equilibrium water content varied between cultivars of both barley and wheat, attributable to agronomic traits. The samples were then inoculated with Penicillium verrucosum and incubated for up to 23 weeks. With time, all cultivars had increasing ochratoxin A content, with maximum content in different barley cultivars ranging from 34 to 630 ng/g grain for aw 0.75, and 39 to 260 000 ng/g for aw 0.85. Corresponding values for the wheat cultivars were 25 to 2 300 ng/g and 650 to 5 200 ng/g. Significant varietal differences in ochratoxin A accumulation were observed for barley (P < 0.0001), attributable to equilibrium water content, amylose content and natural ochratoxin A, and for wheat (P < 0.0001), attributable to protein content and natural ochratoxin A. Barley ‘SW 1306 95/1203’ and ‘SW 906129 Waxy’, and wheat ‘SW 39103’ accumulated significantly less ochratoxin A than the other cultivars.  相似文献   

14.
Barley (Hordeum vulgare L.) is a cereal grown for animal feed, human consumption, and malting. Nutrient concentrations are important as they provide information regarding the dietary values of barley consumed by animals or human beings. In addition, grain nutrient removal may be useful for refining fertilizer recommendations. A study was conducted in 2015 and 2016 investigating the cultivar effects on grain yield, quality, and grain nutrient concentrations and removal under irrigated conditions for two-row barley cultivars. Adjunct and feed cultivars produced the highest yields compared with the all-malt and food cultivars. Specific quality and nutrient values were greater than or equal to in the food cultivar compared to the malt or feed cultivars. Variations in nutrient concentrations were measured among the adjunct and all-malt cultivars, which could potentially affect the malting and brewing qualities. Grain yield, quality, nutrient concentrations and nutrient removal varied among cultivars grown under identical environmental conditions, which may influence end-use.  相似文献   

15.
One tomato ( Lycopersicon esculentum) gene, LAT52, has been proved to be a suitable endogenous reference gene for genetically modified (GM) tomato detection in a previous study. Herein are reported the results of a collaborative ring trial for international validation of the LAT52 gene as endogenous reference gene and its analytical systems; 14 GMO detection laboratories from 8 countries were invited, and results were finally received from 13. These data confirmed the species specificity by testing 10 plant genomic DNAs, less allelic variation and stable single copy number of the LAT52 gene, among 12 different tomato cultivars. Furthermore, the limit of detection of LAT52 qualitative PCR was proved to be 0.1%, which corresponded to 11 copies of haploid tomato genomic DNA, and the limit of quantification for the quantitative PCR system was about 10 copies of haploid tomato genomic DNA with acceptable PCR efficiency and linearity. Additionally, the bias between the test and true values of 8 blind samples ranged from 1.94 to 10.64%. All of these validated results indicated that the LAT52 gene is suitable for use as an endogenous reference gene for the identification and quantification of GM tomato and its derivates.  相似文献   

16.

The effect of different Cr and Zn concentrations in the soil on the development of Albares wheat and Pedrezuela barley plants at the physiological, biochemical, and structural levels was evaluated during the crop cycle in a greenhouse assay, as well as their potential use in phytoremediation strategies. The accumulation of Cr and Zn in plants was dose-dependent for both cultivars. The highest contents were found in root and the lowest in grain. In the Cr treatments, the decrease with respect to the control in the biomass, relative water content (RWC), chlorophyll content (Chl), and chlorophyll fluorescence values (Fv/Fm) was more pronounced in wheat than in barley. For the Zn treatments, the behavior was the opposite. Barley showed less tolerance to Zn concentrations although its higher translocation factor (TF) and greater biomass make this plant adequate to use in phytoremediation process in soil contaminated with Zn. The electron microscopy studies showed evidence that treatment with both Cr and Zn produced alterations in the cellular ultrastructure of the plant leaves. Cr and Zn induced the production of malondialdehyde (MDA) in both cultivars; the highest concentrations were observed in barley leaves. In general, the ascorbate peroxidase activity (APX) was higher in the plants exposed to metal treatments. The catalase activity (CAT) showed a different behavior depending on the metal studied. These results highlight the potential capacity of Albares wheat for use in the phytoremediation of soils contaminated by Zn and of Pedrezuela barley for use in Cr- and Zn-contaminated soils.

  相似文献   

17.
Several countries have introduced mandatory labeling requirements on foods derived from genetically modified organisms. Real-time quantitative Polymerase Chain Reaction (PCR) has quickly become the method of choice in support of these regulations and requires the development of separate PCR assays targeting the transgenic sequence as well as a specific endogenous gene sequence. To develop a Brassica napus-specific PCR assay, partial sequences of the acetyl-CoA carboxylase BnACCg8 gene from B. napus and the closely related Brassica rapa were determined and compared, and a region of unique nucleotide sequence was identified. Universal amplification primers were designed to either side of this region, and a locked nucleic acid TaqMan probe was designed to the B. napus-specific sequence. Evaluation of this primer/probe combination indicated a high level of specificity to B. napus: no amplification signal was observed with any other species tested, including five closely related Brassica species. The method was assayed with 14 different B. napus cultivars, and comparable amplification curves were consistently obtained for all. The assay was highly sensitive, with a limit of detection between 1 and 10 haploid copies. Practically, the method was demonstrated to be effective for the detection of processed food samples and for the quantification of Roundup Ready canola content in mixed samples.  相似文献   

18.
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   

19.
Genomics is providing a new set of tools for cereal chemistry. Analysis of the DNA of the major cereals is increasing the understanding of the basis of grain quality at the gene level. Differences in the sequence of the gene in different cultivars or differences in the level of expression of the gene may be used to explain differences in processing or end-use quality characteristics. For example, quantitative analysis of the levels of expression of genes at different stages during seed development in wheat or germination in barley can be used to define the genetic basis of differences in wheat and barley quality. Rice quality traits such as fragrance and gelatinization temperature (cooking temperature) can be explained by DNA sequence differences in specific genes identified using genomics approaches. Rapid and reliable species and cultivar identification based on DNA analysis methods developed using genomics tools can be applied to grain and to food products. This technology has special advantages in the analysis of complex mixtures of cereals. Technologies for very high-throughput and very low-cost analysis of large numbers of samples are now available. These may be applied by cereal chemists at many levels: selection in cereal breeding, optimizing processing, and analysis of the identity and composition of grain or food samples.  相似文献   

20.
A simple, rapid method for determining total anthocyanins was developed for use in developing wheat cultivars with dark-blue grains. The method was evaluated as a screening test and for quantification of total anthocyanins in blue and purple wheats and related cereals. Wheat anthocyanins were significantly more extractable in ethanol or methanol than in water at different pH levels. A sample-to-solvent ratio of 1:8 at pH 1 and 25°C was used. Anthocyanin extracts of pigmented wheat and barley grains exhibited absorbance spectra similar to cyanidin 3-glucoside. The absorbance of anthocyanin extracts of 160 blue wheat experimental lines were significantly correlated with whole-grain Hunterlab color values. Total anthocyanins averaged 157 mg/kg in blue wheat whole meal and 104 mg/kg in purple wheat whole meal, whereas blue wheat bran contained 458 mg/kg as compared with 251 mg/kg in purple wheat bran.  相似文献   

设为首页 | 免责声明 | 关于勤云 | 加入收藏

Copyright©北京勤云科技发展有限公司  京ICP备09084417号