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1.
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   

2.
Many countries have introduced mandatory labeling requirements on foods derived from genetically modified organisms (GMOs). Real-time quantitative polymerase chain reaction (PCR) based upon the TaqMan probe chemistry has become the method mostly used to support these regulations; moreover, event-specific PCR is the preferred method in GMO detection because of its high specificity based on the flanking sequence of the exogenous integrant. The aim of this study was to evaluate the use of very short (eight-nucleotide long), locked nucleic acid (LNA) TaqMan probes in 5'-nuclease PCR assays for the detection and quantification of GMOs. Classic TaqMan and LNA TaqMan probes were compared for the analysis of the maize MON810 transgene. The performance of the two types of probes was tested on the maize endogenous reference gene hmga, the CaMV 35S promoter, and the hsp70/cryIA(b) construct as well as for the event-specific 5'-integration junction of MON810, using plasmids as standard reference molecules. The results of our study demonstrate that the LNA 5'-nuclease PCR assays represent a valid and reliable analytical system for the detection and quantification of transgenes. Application of very short LNA TaqMan probes to GMO quantification can simplify the design of 5'-nuclease assays.  相似文献   

3.
As the genetically modified organisms (GMOs) labeling policies are issued in many countries, qualitative and quantitative polymerase chain reaction (PCR) techniques are increasingly used for the detection of genetically modified (GM) crops in foods. Qualitative PCR and TaqMan real-time quantitative PCR methods to detect and identify three varieties of insect resistant cotton, i.e., Mon531 cotton (Monsanto Co.) and GK19 and SGK321 cottons (Chinese Academy of Agricultural Sciences), which were approved for commercialization in China, were developed in this paper. Primer pairs specific to inserted DNAs, such as Cowpea trypsin inhibitor (CpTI) gene of SGK321 cotton and the specific junction DNA sequences containing partial Cry1A(c) gene and NOS terminator of Mon531, GK19, and SGK321 cotton varieties were designed to conduct the identified PCR assays. In conventional specific identified PCR assays, the limit of detection (LOD) was 0.05% for Mon531, GK19, or SGK321 in 100 ng of cotton genomic DNA for one reaction. Also, the multiplex PCR method for screening the three GM cottons was also established, which could save time and cost in practical detection. Furthermore, a real-time quantitative PCR assay based on TaqMan chemistry for detection of insect resistant gene, Cry1A(c), was developed. This assay also featured the use of a standard plasmid as a reference molecule, which contained both a specific region of the transgene Cry1A(c) and an endogenous stearoyl-acyl carrier protein desaturase (Sad1) gene of the cotton. In quantitative PCR assay, the quantification range was from 0.01 to 100% in 100 ng of the genome DNA template, and in the detection of 1.0, 3.0, and 5.0% levels of three insect resistant cotton lines, respectively, all of the relative standard deviations (RSDs) were less than 8.2% except for the GM cotton samples with 1.0% Mon531 or GK19, which meant that our real-time PCR assays involving the use of reference molecule were reliable and practical for GM insect resistant cottons quantification. All of these results indicated that our established conventional and TaqMan real-time PCR assays were applicable to detect the three insect resistant cottons qualitatively and quantitatively.  相似文献   

4.
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   

5.
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   

6.
三种对虾病毒多重实时荧光PCR检测方法的建立   总被引：1，自引：0，他引：1  
根据基因库中白斑综合征病毒（WSSV）、传染性皮下及造血器官坏死病毒（IHHNV）和桃拉综合征病毒（TSV）的基因序列,设计了WSSV 、IHHNV和TSV的三对特异性引物和三条用不同荧光基团标记的TaqMan探针。对反应条件和试剂浓度进行优化,建立了能够同时检测WSSV 、IHHNV和TSV的三重实时荧光PCR方法。该方法特异性好,对WSSV 、IHHNV和TSV的检测敏感性分别达到20000、20和20000个模板拷贝数;此外抗干扰能力强,对WSSV 、IHHNV和TSV不同模板浓度进行组合,仍可有效地同时检测这三个病毒。对保存的45份经常规PCR检测仅为WSSV 、IHHNV和TSV阳性的样品进行二重实时荧光PCR检测,结果都为阳性,其中2份为WSSV和IHHNV混合感染。本研究建立的三重实时荧光PCR方法用于WSSV、IHHNV和TSV的检测具有特异、敏感、快速、定量等优点。  相似文献   

7.
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.  相似文献   

8.
Hazelnuts (Corylus avellana) are used widely in the food industry, especially in confectionery, where they are used raw, roasted, or in a processed formulation (e.g., praline paste and hazelnut oil). Hazelnuts contain multiple allergenic proteins, which can induce an allergic reaction associated with symptoms ranging from mild irritation to life-threatening anaphylactic shock. To date, immunochemical (e.g., ELISA or dipstick) and PCR-based analyses are the only methods available that can be applied as routine tests. The aim of this study is to make a comparative evaluation of the effectiveness of ELISA and real-time PCR in detecting and correctly quantifying hazelnut in food model systems. To this end, the performances of two commercial ELISAs were compared to those of two commercial and one in-house-developed real-time PCR assays. The results showed that although ELISA seemed to be more sensitive compared to real-time PCR, both detection techniques suffered from matrix effects and lacked robustness with regard to food processing. As these impacts were highly variable among the different evaluated assays (both ELISA and real-time PCR), no firm conclusion can be made as to which technique is suited best to detect hazelnut in (processed) food products. In this regard, the current lack of appropriate DNA calibrators to quantify an allergenic ingredient by means of real-time PCR is highlighted.  相似文献   

9.
从GenBank中获取新型甲型H1N1流感病毒(2009年流行株)HA和NA基因序列,通过多重序列比对之后,设计合成H1和N1基因特异性的引物和探针,用来建立新的甲型H1N1亚型流感病毒的多重实时RT-PCR检测方法.合成的两条荧光探针分别标记FAM和CY5荧光报告基团,荧光淬灭均使用BHQ基团.多重实时RT-PCR实验在ABl7500实时PCR仪上进行,经过40个循环的扩增之后,阳性对照出现标准的S型曲线,并且具有良好的特异性,可以很好地将新的甲型H1N1与传统的甲型H1N1以及H3N2、H5N1、H6N2和H9N2等流感病毒区分.多重实时RT-PCR可以检测到10个拷贝的模板,灵敏度接近检测方法的极限.检测时间短,从加样到反应结束只需要90 min.在对243例发热病人临床样品检测过程中发现7例阳性,1351份猪临床样品检测中未发现阳性,该检测结果和采用中国检验检疫科学研究院研制的试剂盒获得的检测结果一致.本研究所建立的快速、准确和高敏感性的多重实时RT-PCR方法非常适用于新型甲型H1N1流感病毒的实验室筛查.  相似文献   

10.
Quenching probe (QProbe) polymerase chain reaction (PCR) is a simple and cost-effective real-time PCR assay in comparison with other real-time PCR assays such as the TaqMan assay. We used QProbe-PCR to quantify genetically modified (GM) soybean (Roundup Ready soybean). We designed event-specific QProbes for Le1 (soy endogenous gene) and RRS (recombinant gene), and we quantified certified reference materials containing 0.1, 0.5, 1, 2, and 5% GM soybean. The TaqMan assay was also applied to the same samples, and the results were compared. The accuracy of QProbe-PCR was similar to that of TaqMan assay. When GM soybean content was 0.5% or more, the relative standard deviations of QProbe-PCR were less than 20%. QProbe-PCR is sensitive enough to monitor labeling systems and has acceptable levels of accuracy and precision.  相似文献   

11.
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   

12.
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   

13.
A fast and quantitative method was developed to detect transgenic "Maximizer" maize "event 176" (Novartis) and "Roundup Ready" soybean (Monsanto) in food by real-time quantitative PCR. The use of the ABI Prism 7700 sequence detection system allowed the determination of the amplified product accumulation through a fluorogenic probe (TaqMan). Fluorescent dyes were chosen in such a way as to coamplify total and transgenic DNA in the same tube. Using real-time quantitative PCR, 2 pg of transgenic or total DNA per gram of starting sample was detected in 3 h after DNA extraction and the relative amounts of "Maximizer" maize and "Roundup Ready" soybean in some representative food products were quantified.  相似文献   

14.
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   

15.
Except for the events RT73, MS8, RF3, and T45, event-specific detection methods for most commercialized genetically modified (GM) rapeseed varieties have not been established, and as a result, the enforcement of genetically modified organism labeling policies has been hindered. The genetically modified rapeseeds, MS1xRF1 and MS1xRF2, are 2 of 11 approved GM-rapeseed varieties for commercialization. In this study, the right border junction fragments between the gene construct and the rapeseed genome of events RF1, RF2, and MS1 were isolated using the commercially available GenomeWalker technology. Homology analysis indicated that the gene construct of RF1 integrated upstream of the nuclease gene, and that of the RF2 and MS1 inserted into the exon region of a gene encoding for an unknown protein. The event-specific primer pairs and corresponding probes were designed on the basis of the revealed right border junction fragments. Then, we successfully developed the identification and quantification methods for the gene-stacked hybrids MS1xRF1 and MS1xRF2 using those primers and probes. The relative limit of detection in the qualitative polymerase chain reaction (PCR) was 0.013% for the RF2 and MS1 assays using 100 ng of rapeseed DNA per reaction and 0.13% for the RF1 assay. The absolute limit of detection in the quantitative PCR was approximately one to two initial copies for each of the three event-specific assays. The evaluation of the real-time PCR assays revealed that the qualitative and quantitative methods developed by focusing on the gene-stacked hybrids MS1xRF1 and MS1xRF2 were highly specific, sensitive, and suitable for samples with a low quantity of DNA.  相似文献   

16.
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   

17.
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   

18.
The genetically modified common bean Embrapa 5.1, developed by Brazilian Agricultural Research Corporation (Embrapa), is the first commercial GM plant produced in Latin America. It presents high resistance to the Bean golden mosaic virus. In this work, primers and probes targeting a taxon-specific reference DNA sequence for the common bean (Phaseolus vulgaris L.) and a construct-specific DNA sequence of Embrapa 5.1 GM common bean were successfully developed. The primers and probes showed high specificity for the target detection. Both methods showed suitable efficiency and performance to be used as an endogenous target for detection of common bean DNA and for construct-specific detection of GM common bean Embrapa 5.1, respectively. Both real-time PCR assays proved to be valuable for future assessment of interlaboratory studies.  相似文献   

19.
Biospecific interaction analysis (BIA) was performed using surface plasmon resonance (SPR) and biosensor technologies to detect genetically modified Roundup Ready soybean gene sequences. We first immobilized, on SA sensor chips, single-stranded biotinylated oligonucleotides containing soybean lectin and Roundup Ready gene sequences, and the efficiency of hybridization to oligonucleotide probes differing in length was determined. Second, we immobilized biotinylated PCR products from nontransgenic soybeans (genomes carrying only the lectin gene), as well as from genetically modified Roundup Ready soybean, and we injected the oligonucleotide probes. Furthermore, we used the sensor chips carrying either lectin and Roundup Ready soybean PCR products or 21-mer oligonucleotide as probes, and we injected both nonpurified and purified asymmetric PCR products. The results obtained show that 13 and 15 mer oligonucleotides are suitable probes to detect genetically modified Roundup Ready soybean gene sequences (either target oligonucleotides or PCR products) under standard BIA experimental conditions. By contrast, when 11 mer DNA probes were employed, no efficient hybridization was obtained. All the SPR-based formats were found to be useful for detection of Roundup Ready gene sequences, suggesting that these procedures are useful for the real-time monitoring of hybridization between target single-stranded PCR products, obtained by using as substrates DNA isolated from normal or transgenic soybeans, and oligonucleotide or PCR-generated probes, therefore enabling a one-step, nonradioactive protocol to perform detection.  相似文献   

20.
Real-time uniplex and duplex polymerase chain reaction (PCR) assays with a SYBR Green I post-PCR melting curve analysis were evaluated for the identification and quantification of bovine, porcine, horse, and wallaroo DNA in food products. Quantitative values were derived from threshold-cycle (C(t)) data obtained from serial dilutions of purified DNA. The limits of detection in uniplex reactions were 0.04 pg for porcine and wallaroo DNA and 0.4 pg for cattle and horse DNA. Species specificity of the PCR products was tested by the identification of peaks in DNA melting curves, measured as the decrease of SYBR Green I fluorescence at the dissociation temperature. The peaks could be distinguished above the background even at the lowest amount of template DNA detected by the C(t) method. The system was also tested in duplex reactions, by use of either single-species DNA or DNA admixtures containing different shares of two species. The minimum proportions of each DNA species allowing the resolution of T(m) peaks in the duplex reactions were 5% (cattle or wallaroo) in cattle/wallaroo mixtures, 5% porcine and 1% horse in porcine/horse mixtures, 60% porcine and 1% wallaroo in porcine/wallaroo mixtures, and 1% cattle and 5% horse in cattle/horse mixtures. A loss in the sensitivity of the method was observed for some DNA combinations in the duplex assay. In contrast, the results obtained from SYBR Green I uniplex and duplex reactions with single-species DNA were largely comparable to those obtained previously with species-specific TaqMan probes, showing the suitability of that simpler experimental approach for large-scale analytical applications.  相似文献   

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