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

2.
根据GenBank中致病性嗜水气单胞菌特异性的气溶素基因序列,设计1对引物,利用普通PCR技术扩增获得hlyA基因片段,并克隆到pMD-18T载体上作为阳性标准品。通过对SYBR GreenⅠ荧光定量PCR反应条件的优化,建立了快速检测致病性嗜水气单胞菌的SYBR GreenⅠ荧光定量诊断方法,以此为基础研制出试剂盒。试剂盒扩增产物的熔解曲线分析只出现1个单特异峰,无引物二聚体,对非致病性嗜水气单胞菌、弗氏柠檬酸杆菌、迟缓爱德华菌、柱状黄杆菌均无阳性信号扩增,重复性好,灵敏度可达1.0x101拷贝/uL。结果表明研制的致病性嗜水气单胞菌SYBR GreenⅠ实时荧光定量PCR试剂盒具有特异、灵敏、快速、重复性好等特点,适合于大鲵临床样品的检测。  相似文献   

3.
We compared ISSR patterns generated by three variants of resolving medium/staining procedure: agarose/ethidium bromide, polyacrylamide/silver staining, and polyacrylamide/SYBR Green I in order to prepare methodology for large-scale screening of genetic diversity in flax (Linum usitatissimum L.) germplasm collections. We found out that polyacrylamide/SYBR Green I system is the most suitable variant for large-scale characterization of genetic diversity within flax germplasm collections.  相似文献   

4.
转植酸酶基因(phyA2)玉米定性、定量PCR检测   总被引：1，自引：0，他引：1  
转植酸酶基因玉米检测是对转植酸酶基因玉米释放进行有效监管的重要前提和技术支撑。本研究根据转植酸酶基因玉米(Zeamays)外源植酸酶基因phyA2序列设计基因特异性引物,进行了定性、定量PCR检测。定性结果显示,该引物可有效检测植酸酶玉米中phyA2基因,而在其他不含植酸酶基因的材料中则检测不到相应基因,表明该引物具有很高的特异性。以玉米内标准基因玉米淀粉合酶异构体zSTSII-2基因(zSSIIb)和植酸酶基因phyA2为对象,进行SYBRGreenⅠ染料法荧光定量PCR反应,绘制2种基因扩增循环数-拷贝数标准曲线图,根据混合样品中(zSSIIb)和植酸酶基因phyA2的拷贝数计算样品中的转基因含量,并做熔解曲线分析。结果表明,zSSIIb和phyA2标准曲线相关系数分别为0.998和0.995,相关性较好,两个基因的熔解曲线均呈单峰型,表明引物特异性较强,混合玉米样品(植酸酶玉米含量分别为1%、0.5%和0.1%)中植酸酶玉米含量分别为1.1%、0.54%和0.17%,实测值与理论值接近。本研究建立了转植酸酶基因玉米定性、定量PCR检测体系,可有效地对转植酸酶基因玉米进行检测。  相似文献   

5.
The present paper describes results of a national survey conducted between 1982 and 1989 to determine residues of arsenic, cadmium, copper, mercury, lead, selenium, and zinc in Canadian slaughter animals. Liver and kidney tissues from cattle, swine, poultry, horses, calves, and sheep were tested. Arsenic was found in most avian and porcine samples, and their respective means of 0.36 and 0.26 micrograms/g in liver were 7 to 12 times higher than mean concentrations found in the other species. Cadmium was found in the tissues of all species; however, levels were consistently highest in equine samples with mean values of 3.09 and 27.7 micrograms/g in liver and kidney, respectively. Copper levels greater than 150 micrograms/g were found predominantly in liver from calves and sheep, with values considerably lower in the remaining species. Mercury levels were low or not detected in all species except horses. Ninety % of equine kidneys and 54% of equine livers had mercury concentrations greater than 0.01 microgram/g, with mean values of 0.18 and 0.06 microgram/g, respectively. Lead was found in tissues of all species; however, values greater than 2 micrograms/g were found only in 2 kidneys from adult cattle and 1 kidney from a horse. Selenium, tested only in cattle, was found at mean concentrations of 0.28 microgram/g in liver, and 0.92 microgram/g in kidney. Relatively high zinc levels were found in livers of horses, pigs, and calves, with respective mean concentrations of 67.3, 65.6, and 70.2 micrograms/g.  相似文献   

6.
This research developed and evaluated a PCR procedure to detect beef in heated and unheated meat, sausages, and canned food, using a specific and sensitive method. To confirm the effectiveness and specificity of this fragment, we tested 45 cattle blood DNA samples (from different breeds) and obtained positive results. With 125 samples tested from other species, the specific beef amplification was not detected. Feed components intended for cattle nutrition were also checked, and bovine-derived material was detected. Using this method we can detect the degree of contamination up to 0.01% raw beef in pork. In the same way, 1% beef was detected in cooked meat mixtures and bovine-derived material in concentrate mixtures. Beef has been identified in both heated and unheated meat products, sausages, canned food, and hamburgers. In conclusion, specific PCR amplification of a repetitive DNA element seems to be a powerful technique for the identification of beef in processed and unprocessed food, because of its simplicity, specificity and sensitivity. Furthermore, feed components intended for cattle nutrition can be checked. The procedure is also much cheaper than other methods based on RFLPs-PCR, immunodiffusion, and other techniques that need expensive equipment.  相似文献   

7.
为确定猪生长激素基因(pGH)在F1代转基因猪体内的遗传稳定性及其在育肥初期表达水平的差异,利用SYBRGreenⅠReal-time PCR法检测精子介导纳米基因载体法获得的F0代母猪与非转基因公猪配种所产的7头转pGH基因猪和9头非转基因猪3月龄生长激素的表达水平。根据16个血液样品荧光定量结果绘制溶解曲线和扩增曲线图谱,并用F=(1+E)﹣ΔΔCt,公式进行Ct均值的分析,得出F1代转pGH基因猪生长激素平均表达水平是同期非转基因猪的1.77倍。结果证实,1)外源pGH基因在转基因猪体内能稳定传代;2)并非每头转基因猪体内pGH基因的表达量均高于非转基因猪;3)在F1代转基因猪体内,pGH的平均表达水平显著高于同期非转基因猪。本实验建立了能够稳定测评家猪生长激素相对基因表达水平的方法,为研究转基因与非转基因动物之间生理指标与定量表达的差异性等提供了科学依据。  相似文献   

8.
中国大菱鲆虹彩病毒(turbot reddish body iridovirus, TRBIV)是一种感染养殖大菱鲆的鱼类虹彩病毒,它可以引起大菱鲆病毒性红体病并导致养殖大菱鲆大量死亡。本研究利用TRBIV主要衣壳蛋白基因序列设计的一对引物,结合内嵌式核酸染料SYBR Green І,建立了TRBIV特异的Real-time PCR检测方法。实验结果表明,该对引物具有较高的灵敏度和较强的特异性,能够检测相当于102数量级的TRBIV基因组拷贝,而不与健康大菱鲆组织DNA、淋巴囊肿病毒DNA发生交叉反应。最后,应用建立的Real-time PCR检测方法,开展了TRBIV的组织敏感性检测和病毒流行情况调查。结果发现,大菱鲆的脾脏、肾脏、脑、鳃、心脏、肝脏、消化道、血液等组织中均可检测到TRBIV的存在,其中脾和肾是TRBIV的最主要的靶器官,每毫克组织的病毒含量分别高达5.23106个和2.18106个。分子流行病学调查结果显示,在山东半岛的多个大菱鲆养殖场中均存在TRBIV的感染和流行。  相似文献   

9.
采用比较基因组学和RT-PCR方法，根据人和小鼠SMAF1基因的保守序列，设计简并引物，克隆了猪SMAF1基因的cDNA序列。所克隆片断全长256bp（GeneBank登录号 DQ191892），包括一个编码了81个氨基酸的完整编码区，该序列与人和小鼠的SMAF1基因的相似性分别为86%和78％，预测的氨基酸序列与人、小鼠、大鼠和牛的相似性分别为81％、67％、70%和84%。猪SMAF1基因在脂肪组织中高丰度表达，在4月龄瘦肉型猪（大白猪）脂肪组织中的表达量显著低于脂肪型猪（梅山猪）（P＜0.05）。本研究结果表明，猪SMAF1基因可能具有和其它物种SMAF1基因相似的功能，推测其在脂肪细胞发生和/或脂肪细胞功能中具有重要的调控作用。  相似文献   

10.
Population viability analysis (PVA) has become a widely used set of tools for evaluating relative extinction risk and prioritizing management options among imperiled populations. While PVA is a widely sanctioned tool in conservation biology, the field of population viability is in its infancy with respect to species interactions. In this paper, I review available methods for evaluating extinction risk when species interactions contribute significantly to population viability. This review includes an evaluation of six broad categories of species interactions (predation, disease, competition, mutualism, parasitism and host-parasitoid interactions) in population viability analysis, with a particular focus on predation as a case study. I first evaluated how often species interactions are considered when PVA is applied to population data from imperiled species. I identified 378 articles in commonly cited conservation journals, of which 24 attempted a viability analysis for populations threatened by interactions with other species. Most of these PVA’s treat a putative species interaction as a constant source of mortality rather than a coupled, dynamic population process. Second, I reviewed the literature to identify the availability of time-series of abundance data for two interacting species in which at least one species was threatened or endangered. Adequate time-series data were available for both species comprising an interacting pair in only 9 out of 407 papers reviewed. Third, I used a stochastic, fully stage-structured predator prey model to create time-series data (vital rates and projection matrices) in order to quantify the efficacy of two matrix-based, single-species PVA approaches. Simple single-species PVAs confound stochastic variation with population cycles induced by species interactions (in this case predation). As a result these models provide conservatively biased forecasts of viability. Unfortunately, the data needed to construct more complex PVA’s with feedback and multi-species stochasticity are rarely collected. I close with a discussion of key advances needed to “escape the population vacuum” in a move toward more realistic estimates of extinction risk.  相似文献   

11.
The primary objective of this study was to test the usefulness of two indices—edge diversity and cover type diversity—in assessing bird species diversity for breeding avian communities in freshwater coastal marshes. Bird species diversity was used in this study as an indicator of ecological quality. Permanent transects in three marshes on the bay of Green Bay, Lake Michigan, provided the basis for vegetation assessments and breeding bird censuses. Four years of field data were subjected to regression analysis. The best equation was found to be a multiple regression of bird species diversity on cover type diversity and edge diversity, showing a significant linear relationship with cover type diversity and a significant curvilinear relationship with edge diversity. Cover type diversity and edge diversity can be readily assessed from aerial imagery. The results are most applicable to ecological evaluation where objectives stress species diversity rather than single-species management.  相似文献   

12.
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   

13.
为建立一种快速准确检测马铃薯卷叶病毒(potato leafroll virus,PLRV)的方法,本研究以PLRV CP(coat protein,CP)基因ORF3保守序列为靶标,建立了PLRV反转录环介导等温扩增(RT-LAMP)检测体系,并对建立的RT-LAMP检测体系的特异性、灵敏度、实际应用效果进行评估。结果表明,建立的PLRV RT-LAMP检测方法,在62℃恒温下扩增50 min,扩增产物中加入双链嵌合荧光染色(SYBR Green Ⅰ),阳性样品颜色变为绿色,阴性样品颜色仍为褐色,可直接目测判断待检测样品是否感染PLRV。该方法只特异性检测PLRV,不与马铃薯X病毒(PVX)、马铃薯Y病毒(PVY)、马铃薯S病毒(PVS)、马铃薯A病毒(PVA)和马铃薯M病毒(PVM)等其他5种马铃薯主要病毒发生交叉反应。此外,建立的PLRV RT-LAMP检测方法的灵敏度较反转录PCR(RT-PCR)方法高100倍。田间样品检测验证,该方法与标准RT-PCR方法符合率达100%。本研究建立的以SYBR Green Ι为颜色指示的PLRV可视化检测RT-LAMP方法,特异、灵敏、便捷,成本低,可满足科研、基层单位对该病毒快速诊断和检测的需要。  相似文献   

14.
This paper describes an assessment of ecological standards for eutrophication and desiccation using the ecotoxicological approach of standard setting. In ecotoxicological risk assessment single-species toxicity data, such as No Observed Effect Concentration (NOEC), are used to extrapolate maximum tolerable concentrations (MTC) which protect 95% of the ecosystem's species. In our approach single-species data consist of the species-response function for both nutrient availability and soil moisture. These data were used to calculate the maximum levels at which 95% of the plant species are protected. A case study, using data on 275 plant species of usable grasslands in the province of South Holland, has been carried out. Ecological standards are calculated for maximum nitrogen load and changes in groundwater level. The influence of nitrogen limitation and moisture shortage on the 95% species protection level is considered. Differences in methodology between our approach and the assessment of ecological standards for micro-pollutants and critical loads are evaluated.  相似文献   

15.
A method of fluorescent Polymerase Chain Reaction-restriction fragment length polymorphism (PCR-RFLP) was applied as an analytical and quantitative tool for meat identification. Following alignments of the nucleotide sequences, an oligonucleotide primer pair was designed to amplify the partial sequences within the 12S ribosomal RNA (12S rRNA) gene of mitochondrial DNA from porcine, caprine, and bovine meats. No fragment can be amplified from dog, cat, fish, duck, goose, turkey, and chicken DNA with the primer pair. Using fluorescence sensor capillary electrophoresis, the species-specific DNA fingerprints of pork, goat, and beef were generated by restriction enzyme digestion following a fluorescence-labeling PCR amplification. Species identification was conducted on the meat mixtures. The reliably semiquantitative levels were below 1% for binary mixtures of pork, goat, and beef. Cooking and autoclaving of meats did not influence the generation of the PCR-RFLP profiles or the analytical accuracy.  相似文献   

16.
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   

17.
Recent studies have demonstrated that mass loss, nutrient dynamics, and decomposer associations in leaf litter from a given plant species can differ when leaves of that species decay alone compared to when they decay mixed with other species’ leaves. Results of litter-mix experiments have been variable, however, making predictions of decomposition in mixtures difficult. It is not known, for example, whether interactions among litter types in litter mixes are similar across sites, even for litter mixtures containing the same plant species. To address this issue, we used reciprocal transplants of litter in compartmentalized litterbags to study decomposition of equal-mass litter mixtures of sugar maple (Acer saccharum Marshall) and red oak (Quercus rubra L.) at four forest sites in northwestern Connecticut. These species differ significantly in litter quality. Red oak always has higher lignin concentrations than maple, and here C:N is lower in oak leaves and litter, a pattern often observed when oak coexists with maple. Overall, we observed less mass loss and lower N accumulation in sugar maple and red oak litter mixtures than we predicted from observed dynamics in single-species litterbags. Whether these differences were significant or not depended on the site of origin of the leaves (P<0.02), but there was no significant interaction between sites of decay and the differences in observed and predicted decomposition (P>0.2) . Mixing of leaf litter types could have significant impacts on nutrient cycling in forests, but the extent of the impacts can vary among sites and depends on the origin of mixed leaves even when the species composition of mixes is constant.  相似文献   

18.
In the present study a technique was developed with the aim of guaranteeing the composition and security of fish meals, since it allows verification of whether these meals contain land animal remains. The method is based on polymerase chain reaction (PCR) and length polymorphism, followed by a restriction fragment length polymorphism (RFLP). Specific primers for every species were designed and calibrated, generating exclusively a PCR product with a specific size when DNA for each species was present in the sample. This technique allows the detection of land animal remains in fish meals, specifically cow, chicken, pig, horse, sheep, and goat. The identity of the PCR products can be confirmed by RFLP analysis using only one restriction enzyme. The selected restrictase generated one characteristic restriction profile for every species included in this study. The detection limit of this method was calculated by using mixtures of fish meals in different proportions and meal that exclusively contained remains of one of these land species studied. The analytical strategy herein proposed was applied to fish and meat meals, giving good results, both in the analyzed standards and in commercial samples.  相似文献   

19.
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.  相似文献   

20.
不同动物部分组织基因组甲基化程度的差异分析   总被引：10，自引：1，他引：10  
应用甲基敏感扩增多态性(MSAP)技术,检测和分析了猪、牛、羊、小鼠、鸡和鸭基因组的甲基化程度。结果表明,对CCGG位点,实验中检测的几种动物的甲基化程度多数在0.40￣0.50之间(不包括牛);不同动物来源相同组织基因组的甲基化程度不同,相同动物不同组织基因组的甲基化模式具有特异性;同一种动物,组织基因组的甲基化程度一般都高于血液基因组;另外,哺乳动物与禽类基因组甲基化程度未发现较大的差别,但哺乳动物基因组全甲基化位点较多,半甲基化位点较少。  相似文献   

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