Qualitative and quantitative PCR methods for detection of three lines of genetically modified potatoes

Qualitative and quantitative polymerase chain reaction (PCR) methods have been developed for the detection of genetically modified (GM) potatoes. The combination of specific primers for amplification of the promoter region of Cry3A gene, potato leafroll virus replicase gene, and potato virus Y coat protein gene allows to identify each line of NewLeaf, NewLeaf Y, and NewLeaf Plus GM potatoes. Multiplex PCR method was also established for the simple and rapid detection of the three lines of GM potato in a mixture sample. For further quantitative detection, the realtime PCR method has been developed. This method features the use of a standard plasmid as a reference molecule. Standard plasmid contains both a specific region of the transgene Cry3A and an endogenous UDP-glucose pyrophosphorylase gene of the potato. The test samples containing 0.5, 1, 3, and 5% GM potatoes were quantified by this method. At the 3.0% level of each line of GM potato, the relative standard deviations ranged from 6.0 to 19.6%. This result shows that the above PCR methods are applicable to detect GM potatoes quantitatively as well as qualitatively.     

Development and evaluation of event-specific qualitative PCR methods for genetically modified Bt10 maize

In 2005 it was reported that the genetically modified (GM) maize strain or "event" called Bt10 had been distributed inadvertently in the United States over the previous 4 years. In order to ensure that grain for food and feed production did not contain trace amounts of Bt10 maize and complied with the applicable regulation, highly sensitive and specific detection of Bt10 maize was required. Accordingly, we developed a novel qualitative PCR system for specific detection of Bt10 maize. Moreover, we amply evaluated the performance characteristics of two PCR systems, our own and the one provided by the developer of Bt10, Syngenta Co. Ltd. It was confirmed that both of the qualitative PCR systems can specifically detect Bt10 maize, and the results of a single-laboratory examination suggested that the limit of detection was approximately less than 0.05% for both methods. To evaluate the reproducibility of the methods, we organized an interlaboratory study with the participation of 6 laboratories and analysis of 240 blind test samples. In this paper, we report, for the first time, the statistical analysis of the qualitative PCR data obtained from the interlaboratory study. The results of this analysis also revealed that there was no significant difference in the sensitivity between the two aforementioned methods and that the limit of detection of both the methods was less than 0.05%. Thus, we conclude that both of the methods are equally suitable for correct identification and sensitive detection of the unapproved GM maize Bt10 event in test samples.     

Event specific qualitative and quantitative polymerase chain reaction detection of genetically modified MON863 maize based on the 5'-transgene integration sequence

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.     

Event-specific qualitative and quantitative PCR detection methods for transgenic rapeseed hybrids MS1xRF1 and MS1xRF2

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.     

Qualitative and quantitative liquid chromatographic analysis methods for the determination of the effects of feed supplements on hen egg yolk phospholipids

Quantitative and qualitative high-performance liquid chromatographic methods were utilized to separate phospholipid classes. After qualitative separation, the fatty acid moieties of each separated phospholipid class were determined using a gas chromatographic method. On the basis of these analyses, the effect of supplemented feeds on hen egg yolk lipids can be evaluated. The supplemented feeds contained 1-5% of vegetable-based or fish oils. The phospholipid content and composition were the same in all feeding groups, the proportions of phosphatidylcholines, phosphatidylethanolamines, and sphingomyelins being 70, 28, and 3%, respectively. In each feeding group, the fatty acid profiles of phosphatidylcholines and sphingomyelins were similar to each other and different from that of phosphatidylethanolamines. The supplemented feeds had a statistically significant (p < 0.05) effect on the fatty acid composition of phosphatidylcholines. The supplements decreased the proportion of saturated fatty acids in total fat, but this effect was not found in phospholipids.     

Event-specific plasmid standards and real-time PCR methods for transgenic Bt11, Bt176, and GA21 maize and transgenic GT73 canola

Since the 18th of April 2004, two new regulations, EC/1829/2003 on genetically modified food and feed products and EC/1830/2003 on traceability and labeling of GMOs, are in force in the EU. This new, comprehensive regulatory framework emphasizes the need of an adequate tracing system. Unique identifiers, such as the transgene genome junction region or a specific rearrangement within the transgene DNA, should form the basis of such a tracing system. In this study, we describe the development of event-specific tracing systems for transgenic maize lines Bt11, Bt176, and GA21 and for canola event GT73. Molecular characterization of the transgene loci enabled us to clone an event-specific sequence into a plasmid vector, to be used as a marker, and to develop line-specific primers. Primer specificity was tested through qualitative PCRs and dissociation curve analysis in SYBR Green I real-time PCRs. The primers were then combined with event-specific TaqMan probes in quantitative real-time PCRs. Calibration curves were set up both with genomic DNA samples and the newly synthesized plasmid DNA markers. It is shown that cloned plasmid GMO target sequences are perfectly suitable as unique identifiers and quantitative calibrators. Together with an event-specific primer pair and a highly specific TaqMan probe, the plasmid markers form crucial components of a unique and straighforward tracing system for Bt11, Bt176, and GA21 maize and GT73 canola events.     

Sampling plan and test protocol for the semiquantitative detection of genetically modified canola (Brassica napus) seed in bulk canola seed

Using a statistical approach, sampling plans for the semiquantitative detection of genetically modified (GM) canola within a bulk seed sample can be developed and tailored to meet different GM thresholds, costs, and confidence limits. This is achieved by changing the number of subsamples analyzed, the number of seeds per subsample, and the percentage of positive results allowed. These sampling plans must be devised carefully, taking into account the detection capability of the analytical assay. This is particularly important in the case of InVigor (a registered trademark of Bayer CropScience) canola, for which expression levels of the introduced protein in seed are very low. Lateral flow assays and enzyme-linked immunosorbent assays (ELISA) were both investigated for their suitability as a qualitative assay using a subsampling approach. On the basis of an ELISA, several sampling plans have been devised and validated to provide at least 99% confidence that bulk seed samples containing at least 0.9% (w/w) InVigor canola will be detected. Although the term "seed" is used throughout this paper to refer to the canola, the term "seed" is to be taken to include both seed and the canola seed (grain) that is harvested by the farmer/grower.     

Qualitative and quantitative evaluation of the genomic DNA extracted from GMO and non-GMO foodstuffs with four different extraction methods

The presence of DNA in foodstuffs derived from or containing genetically modified organisms (GMO) is the basic requirement for labeling of GMO foods in Council Directive 2001/18/CE (Off. J. Eur. Communities 2001, L1 06/2). In this work, four different methods for DNA extraction were evaluated and compared. To rank the different methods, the quality and quantity of DNA extracted from standards, containing known percentages of GMO material and from different food products, were considered. The food products analyzed derived from both soybean and maize and were chosen on the basis of the mechanical, technological, and chemical treatment they had been subjected to during processing. Degree of DNA degradation at various stages of food production was evaluated through the amplification of different DNA fragments belonging to the endogenous genes of both maize and soybean. Genomic DNA was extracted from Roundup Ready soybean and maize MON810 standard flours, according to four different methods, and quantified by real-time Polymerase Chain Reaction (PCR), with the aim of determining the influence of the extraction methods on the DNA quantification through real-time PCR.     

Qualitative and quantitative land-suitability evaluation for sunflower and maize in the north-west of Iran

Land evaluation is the act of predicting the use potential of land on the basis of its attributes. The objective of this study was to evaluate qualitative and quantitative land suitability for the north-west of Iran on the basis of a FAO model for sunflower and maize crops. Growing cycle was determined using the CDBm (Monthly Climate Database) model and soils were classified in two orders of Mollisols and Vertisols, which consisted of seven soil families based on soil data and Keys to Soil Taxonomy 2010. Qualitative evaluation was carried out using the square root of parametric (SRP) method and quantitative evaluation was performed on the basis of observed yields under an average management level. The results showed that in the surveyed area, the climatic class had moderate suitability (S2) for maize and sunflower due to limitations imposed by the relative humidity of the growing cycle. The most important land limitation factors were soil parameters like pH, CaCO₃ content, texture and coarse fragments, as well as topography and drainage in the area of study. Based on qualitative evaluation with SRP, 24.69 and 17.71% of land had non-suitable class (N1) for maize and sunflower, respectively. Quantitative land suitability for maize and sunflower showed that 24.69 and 9.81% of land had non-suitable class (N1), respectively.     

基于3S和实测相结合的冬小麦估产研究

该文运用3S集成技术（地理信息系统、遥感和全球定位系统）,进行冬小麦产量估测的应用研究。结果表明, 3月下旬是运城地区冬小麦面积监测的最佳时相,4月上、中旬是该区冬小麦产量估算的最佳时期。用遥感信息中的TM提取冬小麦面积,AVHRR提取绿度信息,效果较好。根据归一化植被指数的大小把冬小麦分为3类,同类麦田在不同区域的实测产量差异很大。分析研究区域自然地理特征和关键期气象资料,以归一化植被指数、极高温度、相对湿度为主因子建立了冬小麦遥感—气象—产量综合模型。     

基于光流法与特征统计的鱼群异常行为检测

鱼类群体行为的异常检测能够为鱼类健康监控与预警提供重要的方法和手段,对研究鱼类行为的机理,提升水产养殖过程中的信息化水平具有非常重要的意义。该文通过计算机视觉和图像处理技术,基于鱼群运动特征统计方法,对鱼群异常行为检测进行研究。利用Lucas-Kanade光流法得到目标鱼群的运动矢量,并对目标运动的行为特征进行统计,得到速度与转角这2个行为特征的联合直方图与联合概率分布。最后,在联合概率分布的基础上,基于标准互信息(normalized mutual information-NMI)和局部距离异常因子(local distance-based outlier factor-LDOF)2种方法对鱼群行为进行异常检测。试验结果表明,2种异常检测方法均达到99.5%以上的准确率。     

Validation of a rice specific gene, sucrose phosphate synthase, used as the endogenous reference gene for qualitative and real-time quantitative PCR detection of transgenes

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.     

Real-time polymerase chain reaction based assays for quantitative detection of barley, rice, sunflower, and wheat

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.     

基于增强Tiny YOLOV3算法的车辆实时检测与跟踪

针对深度学习方法在视觉车辆检测过程中对小目标车辆漏检率高和难以实现嵌入式实时检测的问题,该文基于Tiny YOLOV3算法提出了增强Tiny YOLOV3模型,并通过匈牙利匹配和卡尔曼滤波算法实现目标车辆的跟踪。在车载Jetson TX2嵌入式平台上,分别在白天和夜间驾驶环境下进行了对比试验。试验结果表明:与Tiny YOLOV3模型相比,增强Tiny YOLOV3模型的车辆检测平均准确率提高4.6%,平均误检率减少0.5%,平均漏检率降低7.4%,算法平均耗时增加43.8 ms/帧;加入跟踪算法后,本文算法模型的车辆检测平均准确率提高10.6%,平均误检率减少1.2%,平均漏检率降低23.6%,平均运算速度提高5倍左右,可达30帧/s。结果表明,所提出的算法能够实时准确检测出目标车辆,为卷积神经网络模型的嵌入式工程应用提供了参考。     

Comparison of methods for the quantitative determination of phospholipids in lecithins and flour improvers

Phospholipid classes were determined qualitatively and quantitatively in eight commercial lecithins and three flour improvers by thin-layer chromatography (TLC), high-performance liquid chromatography (HPLC), and (31)P nuclear magnetic resonance spectroscopy ((31)P NMR). The total amounts of phospholipids as well as the amounts of phospholipid classes in the samples were comparable but depended on the method used for quantification. Highest selectivity was provided by (31)P NMR as all phospholipids and lysophospholipids could easily be quantified. By TLC only lysophosphatidylcholine could not be quantified, whereas HPLC was the method with the lowest selectivity, because lysophospholipids, except lysophosphatidylethanolamine, could not be determined. Sensitivity was best for HPLC and TLC with detection limits of 20-170 mug/mL. By means of (31)P NMR these figures increased by a factor of 10-70. The coefficients of variation were 5.5, 6.8, and 12.8% for quantification by TLC, HPLC, and (31)P NMR, respectively, showing that TLC was the method with the best reproducibility. Altogether, (31)P NMR can be recommended for the quantification of phospholipids, because it is easy to perform and results can be obtained quickly. As it requires minimum instrumental equipment, TLC is a good alternative to (31)P NMR. If high sensitivity is required, HPLC is the best method.     

基于电子鼻的花生有害霉菌种类识别及侵染程度定量检测

针对花生霉变传统分析方法操作繁琐、时效性差等不足,该研究拟利用电子鼻气体传感技术建立起花生有害霉菌污染的快速检测方法。辐射灭菌花生籽粒分别接种5种谷物中常见有害霉菌(黄曲霉3.17、黄曲霉3.395 0、寄生曲霉3.395、寄生曲霉3.012 4和赭曲霉3.648 6),并于26℃、80%相对湿度条件下储藏9 d至严重霉变。利用电子鼻气体传感器获取不同储藏时期(0、3、6、9 d)花生样品的整体挥发性气味信息。最后,结合多元统计分析方法对电子鼻传感器响应信号进行特征提取,建立了花生中有害霉菌污染程度的定性定量分析模型。结果显示,主成分分析法(principal component analysis,PCA)可成功区分不同霉菌侵染程度的花生样品,线性判别分析(linear discriminant analysis,LDA)模型对样品不同储藏天数判别的准确率均达到或接近100%。花生中菌落总数的偏最小二乘回归分析(partial least squares regression,PLSR)模型的预测决定系数和预测相对均方根误差分别达到0.814 5和0.244 0 lg(CFU/g)。结果表明,应用电子鼻技术快速检测储藏期间花生霉变状况具有一定可行性,可为利用气味信息实现粮食霉菌污染的在线监测提供理论参考。     

Toward metrological traceability for DNA fragment ratios in GM quantification. 1. Effect of DNA extraction methods on the quantitative determination of Bt176 corn by real-time PCR

An international CCQM-P60 pilot study involving eight national metrological institutes was organized to investigate if the quantification of genetically modified (GM) corn powder by real-time PCR was affected by the DNA extraction method applied. Four commonly used extraction methods were compared for the extraction of DNA from a GM Bt176 corn powder. The CTAB-based method yielded the highest DNA template quantity and quality. A difference in the 260 nm/230 nm absorbance ratio was observed among the different extraction methods. Real-time amplification of sequences specific for endogenous genes zein and hmg as well as transgenic sequences within the cryIA(b) gene and a fragment covering the junction between the transformed DNA and the plant genome were used to determine the GM percentage. The detection of the transgenic gene was affected by the quantity and quality of template used for the PCR reaction. The Bt176 percentages measured on diluted or purified templates were statistically different depending on the extraction method applied.     

基于红外热成像与改进YOLOV3的夜间野兔监测方法

随生态改善,野兔数量增多,对农田与林地的危害日益加重。野兔活动多为夜间,目标小,运动速度快,且出现环境较复杂,监控兔害,需要一种高效智能化的方法。针对野兔活动习性,该文提出了使用红外热成像实时监控,结合改进的YOLOV3目标检测方法对夜间野兔进行检测。根据YOLOV3目标检测网络基本结构提出了一种针对红外图像中野兔的实时检测的网络(infrared rabbit detection YOLO,IR-YOLO),该网络特征提取部分压缩YOLOV3特征提取网络深度,利用浅层卷积层特征以提高低分辨率红外小目标检测精度,降低运算量,网络检测部分使用基于CenterNet结构的检测方式以提高检测速度。使用热成像野外实时采集的夜间野兔图像作为数据集,包括不同距离,尺度,出现环境不同的野兔共计6 000幅红外图像制作训练集与测试集,比例为5:1。试验结果表明,IR-YOLO在红外热成像视频中复杂环境下出现的野兔检测率达75%,平均检测速度51帧/s,相对改进前YOLOV3检测率提高15个百分点,相对改进前YOLOV3检测速度提高5帧/s。相比其他目标检测算法各项检测指标更为优良,检测率方面相对Faster-RCNN与RFCN-RESNET101分别提高45个百分点与20个百分点,检测速度方面相对Faster-RCNN与RFCN-RESNET101分别提高30和与45帧/s。该方法可高效快速地对夜间复杂环境下出现的野兔进行检测,也可广泛应用于夜间对其他类型农业害兽的检测。     

根区土壤氧气检测技术与方法研究进展

根区氧胁迫问题在滞涝区域、城市地面硬化区域尤为常见,根区土壤氧气含量高低是判断植物是否承受低氧胁迫的重要依据,其检测技术与分析方法的研究对农林生产和生态保护具有指导意义。为明确根区土壤氧气检测现状及发展方向,该研究概括了国内外土壤氧气分布检测方法和分析策略,将现有获取根区土壤氧含量时空分布的技术手段归纳为氧传感器点位检测法、土壤氧扩散模型分析法和荧光成像法,并剖析比较了各方法的优缺点和适用场景。基于氧传感器等领域最新进展,结合各类氧气运输模型,总结了根区土壤氧气分布检测技术和建模方法的研究重点与发展方向如下：1）氧传感器呈现小型化和集成化趋势,引入新型材料,提高氧传感器的灵敏度和耐久性能;2）模型的精度将不断提高,更多地考虑多尺度和多因素交互作用,以全面分析根区土壤氧环境的复杂性;3）开发可持续的荧光染料,提高荧光成像技术的分辨率和深度,以实现对更小、更深层次结构的清晰成像;4）分析植物蒸腾作用中的水氧关系,通过茎干氧浓度反映植物对根区氧环境变化的生理适应能力。研究可为植物根区土壤氧气分布检测提供思路与借鉴。     

A rapeseed-specific gene, acetyl-CoA carboxylase, can be used as a reference for qualitative and real-time quantitative PCR detection of transgenes from mixed food samples

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.