食物或食物原材料中特定属的植物的定量PCR检测方法

一种根据PCR技术测定食物或食物原材料中的特定属的植物量的方法，包括：（1）提供修正样品，其中来自作为检测靶的特定属的植物样品和标准植物样品的混合比率预先确定，并从该样品中提取基因组DNA；（2）通过向作为试验受试者的食物或食物原材料中添加已知量的标准植物样品来制成测试样品，并从该样品提取基因组DNA；（3）用引物和这些基因组DNA实施定量。     

荧光标记DNA高分辨电感耦合等离子质谱定量分析

建立了基于磷元素测量的高分辨电感耦合等离子质谱定量荧光标记DNA的分析方法,该方法定量测量结果可以溯源到国际基本单位（SI）。采用柱层析、超速离心、透析的技术对样品进行纯化,用芯片电泳和电导率测试仪对其进行了纯度检验。然后利用优化后的微波消解方法对荧光标记DNA样品进行了消解处理。从射频功率、等离子气流速、辅助气流速、雾化气流速、采样深度、获取时间等方面对高分辨电感耦合等离子质谱测量条件进行了优化,从物理性干扰、内标、同位素、元素形态等方面对测量进行了校正。利用优化后的方法对荧光标记DNA样品进行了定量测量,从方法的精密度、标准物质、样品称量、标准和样品稀释等方面进行了定量测量结果不确定度的评估。测量结果的扩展不确定度为8.28%(k=2),远优于现在常规的紫外、荧光、色谱测量核酸含量的不确定度。该方法可用于核酸含量标准物质的定值分析。     

肌酐纯度标准物质的定值方法及其不确定度评定研究

通过定性及定量分析,研究了肌酐纯度标准物质的定值方法,并进行了定值分析的不确定度评定。首先使用三重四极杆质谱仪及核磁共振谱仪(氢谱)对肌酐样品进行定性分析,然后采用质量平衡法(包括液相色谱法、水分、灰分、挥发性物质和无机元素分析)与定量核磁共振法共同对肌酐纯度标准物质进行准确定值,最后对定值结果进行不确定度评定。肌酐的定值结果为99.7%,扩展不确定度为0.4%。该研究对于实际检测中肌酐的准确测定及临床上相关疾病的正确诊断治疗具有重要意义,且经过定值的肌酐纯品还可做定量核磁共振法的定量内标使用。定量分析后的肌酐经过均匀性检验和稳定性考察后可申报为国家标准物质。     

能量色散X射线荧光光谱法检测塑料薄片中的铅

针对我国尚无X射线荧光光谱法测量薄片样品中铅含量的现状,使用美国国家标准与技术研究院制造的薄片系列标准物质建立铅含量标准曲线,将已定值的塑料颗粒熔融制作成0.230 mm的薄片样品,检测其中的铅含量,验证本实验方法的准确度和精密度。通过参考块状样品检测的参数选择,建立薄片样品测试条件,研究了薄片检测中饱和厚度、计数时间、含氯基体的影响,并分析其产生原因,提出了应对措施。结果显示使用98.7 mg/kg薄片样品进行5次测量的结果为100.8 mg/kg,精密度为7.4%,相对标准误差为2.1%,能够满足实验室检测的精度需求。本实验方法可以作为薄片样品检测的参考,并对薄片标准物质的生产有参考意义。     

分子生物学手段检测转Bt基因棉花中Cry杀虫蛋白的定性和定量方法

建立了转Bt基因棉花中Cry杀虫蛋白的提取、样品前处理以及酶联免疫(ELISA)定量分析方法,并使用凝胶电泳、普通聚合酶链式反应(PCR)和实时荧光定量PCR等分子生物学手段对转基因棉花中的Bt基因进行定性和定量检测.所建立的苏云金芽孢杆菌杀虫晶体蛋白(Cry1Ab蛋白和Cry1Ac蛋白)标准曲线线性关系良好,相关系数r2均大于0.999,相对标准偏差RSD均小于2.0%.方法简单、快速、重现性和精密度好,可为农业食品行业和环境领域科研人员提供一种简便快速地从转基因棉花中检测Bt毒蛋白的分析方法.     

土壤中有机氯农药检测能力验证结果分析

以山东省市场监督管理局2021年土壤中有机氯农药能力检测验证计划实施过程为例,介绍能力验证计划的设计、实施、结果统计与分析方法。采用国际通用的稳健统计分析方法评判各参加实验室的测试结果,客观反映各参加实验室的测试能力和水平。通过Z比分数分布及原始记录帮助实验室分析存在的检测技术问题,提升实验室检测能力水平。该次能力验证共回收检测结果296个,其中满意结果239个,满意率约80.7%;可疑结果52个,占总数的17.6%;离群结果5个,占比为1.7%。结果表明大多数参加实验室能够准确检测土壤中有机氯农药,部分实验室在标准物质使用、样品处理、人员操作和设备使用方面存在不足,需要根据本次验证结果加以改进。     

同位素稀释的气相色谱／高分辨质谱联用测定食品中二嗯英和共平面多氯联苯

采用HRGC／HRMS和同位素稀释定量技术对样品中17种4～8个氯原子取代的二嗯英和呋喃(PCDDs／Fs)与12种共平面多氯联苯(PCBs)定量分析。样品经索式抽提、FMS Power Prep系统净化、浓缩，利用高分辨气相色谱／高分辨质谱联用仪的多离子检测方式，同位素稀释技术对样品中的目标化合物进行定性和定量。该方法的检出限为pg／g水平。^13同位素内标回收率范围为47％～100％。对3个CRM鱼样中17个PCDDs／Fs和4个PCBs的检测值均在标准定值允许误差范围内。对5个不同的实际样品鱼进行测定表明，样品的回收率在48％～100％之间，回收率的相对标准偏差小于20％；对同一样品进行定量检测的精密度测试结果表明，17种PCDDs／Fs浓度的RSD低于16％，12种PCBs浓度的RSD低于11％。本方法定量分析重现性良好。     

标准物质在检测实验室质量控制中的应用

【】 本文介绍了检测中使用标准物质进行质量控制的重要意义,分别在盲样测试、期间核查和质量控制图应用方面提出了使用标准物质进行质量控制的三种方法,并通过在实验室获得的检测数据对上述应用方法作了实例说明,对检测实验室应用标准物质进行质量控制工作具有指导意义。     

Taqman-MGB实时荧光PCR定量检测转基因玉米MON863

运用实时荧光聚合酶链式反应技术(polymerase chain reaction,PCR)对转基因玉米MON863进行品种特异性检测和定量分析。通过设计玉米内源基因和外源基因边界序列特异性引物和Taqman-MGB探针,验证了内源基因的物种特异性和外源基因边界序列的品种特异性。利用已知转基因百分含量的MON863玉米作为标准品,进行荧光定量反应,建立定量标准曲线,通过标准曲线对玉米样品MON863玉米成份进行含量分析。结果表明,该方法重复性好,检测特异性强,最低检测限浓度达到0.001 ng/μL,即14个拷贝。由于使用实时荧光PCR技术,检测周期短,操作简便,可广泛运用于转基因玉米MON863的进出口检测和转基因产品的含量分析。     

乳液中氢醌的基体标准物质制备

介绍乳液中氢醌基体标准物质制备过程。在阴性乳液样品中加入氢醌标准品，经混匀后分装，得到乳液中氢醌基体标准物质。采用高效液相色谱法测定乳液样品中氢醌的含量，并对样品进行了稳定性检验、定值和不确定度分析，结果均满足标准要求。经3家有资质的实验室联合定值，乳液中氢醌基体标准物质标准值为108μg/g，扩展不确定度为9.86μg/g (k=2)。该标准物质满足基体标准物质要求，可用于乳液中氢醌检测的质量控制。     

食品毒理学测试能力验证的研究

介绍了CNALT0158“食品添加剂毒性测试能力验证计划”,其中包括测试项目的选定、测试样品的制备及测试样品均匀性、稳定性检测方法,并对参试实验室测试结果进行了统计评价。影响半数致死量(LD50)的因素有很多,对于CNALT0158计划中LD50测试结果可能产生显著影响的因素是:实验动物的健康状况、试验环境温度的控制、受试动物的分组等。     

Rapid and Sensitive Detection of <Emphasis Type="Italic">sFAT-1</Emphasis> Transgenic Pigs by Visual Loop-Mediated Isothermal Amplification

Genetically modified (GM) livestock have the potential to contribute to improving the environment and human health, with consumption of fewer resources and reduced waste production. However, the transgene process also poses risks. The safety assessment and control of transgenic animal products have drawn wide attention, and the relevant regulations and technology are being developed. Quick testing technology plays a significant role in on-site and customs sampling. Nowadays, loop-mediated isothermal amplification (LAMP) was widely applied in nucleic acid analysis because of its simplicity, rapidity, high efficiency and specificity. In this study, a specific, sensitive detection system for detecting sFAT-1 transgenic pigs was designed. A set of six primers including two loop primers was designed for the target sequence. The DNA samples were amplified in less than 1 h at the optimized temperature and detecting by both Nephelometer LA-320c and unaided eyes directly adding calcein. The detection limit of sFAT-1 LAMP was as low as 1.26 ng/μL. Furthermore, blind tests of transgenic and non-transgenic DNA samples were all correctly detected. Hence, the results in this study demonstrated that LAMP is a very useful tool for transgenic detection.     

Proficiency testing as a tool to assess the performance of visual TLC quantitation estimates

The use of rapid and inexpensive nonlaboratory-based screening tests for drug quality assessments is recommended as a component of a drug quality assurance program in poor resource settings. We have established routine Minilab test procedures to screen product quality and a proficiency testing program to determine the competency of the inspectors and reliability of results. Samples for the proficiency testing were prepared by pulverizing a standard reference tablet of the appropriate drug and making serial dilutions with starch to obtain concentrations of 0, 40, and 100%. The samples, which were labeled only with the drug name and an identifying letter, were given to inspectors for quality screening using Minilab procedures. In round 1 of the proficiency test, only 3 of 28 substandard samples were correctly identified. Round 2 of the proficiency test, which was administered after a performance qualification test for the analytical method, showed much improvement: 19 of 27 substandard drugs were correctly identified, while 5 out of 9 inspectors made the correct inference on the quality of 45 samples. However, in both rounds, 2 inspectors failed to identify substandard samples, indicating that their technical competencies need to be improved for the reliability of the results. Although the thin-layer chromatography screening methods provide a rapid means for drug quality assessment, they need to be put in the hands of competent users. The inclusion of a proficiency test in the screening program provides a measure of determining competency of the personnel and reliability of the results.     

Detection of genetic modification in cured tobacco leaf: proficiency testing using polymerase chain reaction-based methods

The Cooperation Centre for Scientific Research Relative to Tobacco (CORESTA; Paris, France) "Task Force Genetically Modified Tobacco-Detection Methods" investigated the performance of qualitative and quantitative methods based on the polymerase chain reaction (PCR) for the detection and quantitation of genetically modified (GM) tobacco. In the 4 successful rounds of proficiency testing, the cauliflower mosaic virus 35S RNA promoter (CaMV 35S) and the Agrobacterium tumefaciens nopaline synthase terminator (NOS) were selected as target sequences. Blind-coded reference materials containing from 0.1 to 5.0% and from 0.15 to 4% GM tobacco were used in 2 rounds of qualitative and quantitative PCR, respectively. Eighteen laboratories from 10 countries participated in this study. Considering all methods and 2 rounds, the different laboratories were able to detect GM tobacco at the 0.1% level in 46 out of 58 tests in qualitative assays. The results of the proficiency test indicate that both end point screening and real-time quantitative methods are suitable for the detection of genetically modified organisms in tobacco leaf samples having a GM content of 0.1% or higher. The CORESTA proficiency study represents a first step towards the interlaboratory evaluation of accuracy and precision of PCR-based GM tobacco detection, which may lead to the harmonization of analytical procedures and to the enhancement of comparability of testing results produced by different laboratories.     

Specific detection of DNA through coupling of a TaqMan assay with surface enhanced Raman scattering (SERS)

We have combined the benefits of a TaqMan assay with surface enhanced Raman scattering (SERS), to generate a novel DNA detection method which provides increased sensitivity, with clear applications for disease identification through clinical testing. Target DNA detection limits by SERS were shown to be lower than conventional fluorescence detection and clinically relevant samples of methicillin-resistant Staphylococcus aureus were detected with high specificity.     

Assessment of genetic purity of bulked-seed of rice CMS lines using capillary electrophoresis

Genetically pure cytoplasmic male sterile line (A-line) is essential to generate pure hybrid seeds in order to harness the yield heterosis in rice. Conventionally, seed purity testing is carried by grow-out test, which has many limitations. Seed purity assessments based on molecular markers reduce the time required for analysis significantly. However, it is very tedious as at least 200–400 seeds/seedlings are needed to be analyzed individually. An assay based on bulked-seed and molecular markers will be an ideal system. Keeping these points in view, in the present study, a co-dominant mitochondrial marker was used to test the purity of bulked parental line (A-line) seed utilizing CE system in a genetic analyzer. The results indicate that this method is very simple, accurate, and can be used to test purity of large number of samples rapidly in a cost-effective way compared to grow-out test and conventional molecular marker analysis.     

Estimation of the minimum uncertainty of DNA concentration in a genetically modified maize sample candidate certified reference material

Homogeneity testing and the determination of minimum sample mass are an important part of the certification of reference materials. The smallest theoretically achievable uncertainty of certified concentration values is limited by the concentration distribution of analyte in the different particle size fractions of powdered biological samples. This might be of special importance if the reference material is prepared by dry mixing, a dilution technique which is used for the production of the new and third generation of genetically modified (GMO) plant certified reference materials. For the production of dry mixed PMON 810 maize reference material a computer program was developed to calculate the theoretically smallest uncertainty for a selected sample intake. This model was used to compare three differently milled maize samples, and the effect of dilution on the uncertainty of the DNA content of GMO maize was estimated as well. In the case of a 50-mg sample mass the lowest achievable standard deviation was 2% for the sample containing 0.1% GMO and the minimum deviation was less than 0.5% for the sample containing 5% GMO.     

广西污水中汞、砷检测能力验证结果分析

利用能力验证计划,对广西全区检验检测机构的污水中汞、砷分析能力进行对比和评价。采用单因子方差对考核样品进行均匀性检测,对全区144家检验检测机构的检测结果进行四分位稳健统计分析,用Z比分值绘制柱状图直观评定各检测机构的测试结果。污水中汞检测结果合格率为77.0%,砷检测结果合格率为84.8%。广西检验机构的汞、砷总体检测能力较强,少数检测机构的检测能力和水平有待提高。     

微孔对HDPE缺口冲击强度及断面形貌特征的影响研究

在-196℃～+23℃的温度范围内,系统测试了微孔发泡和未发泡高密聚乙烯(HDPE)的Izod缺口冲击强度,进行了动态粘弹谱(DMA)和冲击断口系统观察分析.根据实验结果,研究了外加冲击力场作用下微发泡高密度聚乙烯变形断裂过程和机理,揭示了微孔的存在导致一定实验温度下的材料变形断裂机制发生了变化,微孔的引入一方面减小了试样(材料)的有效承载面积,另一方面导致HDPE试样芯部基体材料的应力状态改变为平面应力状态,易于在冲击载荷下产生塑性变形或在低温脆断条件下裂纹尖端钝化阻止裂纹扩展,其综合作用的结果导致微孔发泡和未发泡HDPE的Izod缺口冲击强度随实验温度的变化规律存在差异,且实验温度高于-35℃时,微孔发泡HDPE的缺口冲击强度低于未发泡的,实验温度低于-35℃后,微孔发泡HDPE的缺口冲击强度高于未发泡的.     

THE EFFECTS OF PHOTOOXIDATION BY PROFLAVINE ON HeLa CELLS—II. DAMAGE TO DNA

Abstract— HeLa cell suspensions, prelabeled with specific [¹⁴C]-nucleosides, were treated with proflavine and irradiated with visible light (400–500 nm). The DNA was isolated from the cells (as well as from the appropriate control cells) and examined for macromolecular and molecular changes. Although the UV absorbance spectrum of DNA from irradiated HeLa cells showed no discernible change, a fluorescence spectrum (excitation/emission: 305/405) indicated a molecular change in the DNA. Isolated DNA samples were hydrolyzed with 90% formic acid and chromatographed. There were no detectable differences between the irradiated and non-irradiated profile (R _f and radioactivity) for both guanine and adenine. However, the chromatograms of thymine and cytosine showed distinct changes. There was a loss of radioactivity in the [¹⁴C]-thymidine labeled samples, while the [¹⁴C]-cytidine labeled samples indicated the formation of a new compound, containing 10% of the radioactivity, running just ahead of cytosine. These data strongly suggest the formation of a new compound resulting from the photooxidation of cytosine when nuclear DNA was sensitized by proflavine.