detection method

检测方法
  • 文章类型: Journal Article
    间变性淋巴瘤激酶(ALK)重排的存在定义了非小细胞肺癌(NSCLC)的分子亚型。ALK抑制剂(ALKIs)在ALK阳性晚期NSCLC患者中具有显著的临床获益;因此,准确选择,快速,和适当的ALK测试方法,以筛选适合抗ALK治疗的患者。近年来,ALKIs的开发和临床应用取得了很大进展,以及我们对获得性耐药机制的理解。同时,新的ALK辅助诊断平台已被开发并应用于临床实践。尽管许多研究表明,这些平台之间的一致性很高,在测试过程中不断出现新的问题。为患者带来最大利益,首先选择合适的测试方法,然后制定,优化,并根据检测人群和标本类型遵守标准化的检测流程。随着临床实践数据的不断积累,ALK检测质量控制的经验,以及多中心研究的结果,更新的专家共识是必要的。参与讨论和制定本指南的专家具有丰富的理论背景和临床检验经验,这确保了本指南中提供的信息的实用价值。
    The presence of anaplastic lymphoma kinase (ALK) rearrangement defines a molecular subtype of non-small cell lung cancer (NSCLC). ALK inhibitors (ALKIs) confer significant clinical benefits in patients with ALK-positive advanced NSCLC; therefore, it is of great clinical significance to select accurate, rapid, and appropriate ALK testing methods to screen for patients who are suitable for anti-ALK treatment. In recent years, great progress has been made in the development and clinical application of ALKIs, as well as in our understanding of acquired drug resistance mechanisms. Meanwhile, new ALK companion diagnostic platforms have been developed and applied in clinical practice. Although many studies have shown that there is a high rate of concordance among these platforms, new problems continue to appear during testing. To maximize the benefit for patients, accurate testing results can be obtained by first selecting the appropriate testing method and then formulating, optimizing, and complying with the standardized testing process in accordance with the testing population and specimen types. With the ongoing accumulation of clinical practice data, experience from quality control of ALK testing, and results from multicenter research, an updated expert consensus is necessary. The experts who participated in the discussion and development of this guideline have a rich background in theoretical and clinical testing experience, which ensures the practical value of the information presented in this guideline.
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  • 文章类型: English Abstract
    在日本官方检测未经授权的转基因(GM)木瓜的方法中,具有DNA聚合酶的两种类型的实时PCR试剂之一(TaqMan基因主混合物[TaqMan基因]或FastGeneQPCR探针Mastermixw/ROX[FastGene])主要用于测量。2022年,我们对未经授权的转基因木瓜品系PRSV-YK进行了实验室性能研究,结果表明,使用TaqMan基因与7500Fast和7500Real-TimePCR系统(ABI7500)和QuantStudio12KFlex(QS12K)获得了PRSV-YK检测测试的高阈值循环(Cq)值,表明假阴性的可能性。需要评估所有未经授权的GM木瓜线检测测试出现类似问题的可能性。在这项研究中,我们对未经授权的转基因木瓜品系(PRSV-YK,PRSV-SC,和PRSV-HN),花椰菜花叶病毒35S启动子(CaM),和木瓜阳性对照(Chy),并检查了每种测试的检测限(LOD)如何受到两种类型的DNA聚合酶(TaqMan基因和FastGene)和三种类型的实时PCR仪器(ABI7500,QS12K,和LightCycler480仪器II[LC480])。在使用ABI7500和QS12K的PRSV-YK和PRSV-SC检测试验中,用TaqMan基因测量显示比FastGene更高的LOD。在这种情况下,在扩增图上证实了指数扩增曲线;然而,扩增曲线没有越过ΔRn阈值线,并且在阈值线=0.2的情况下没有获得正确的Cq值。其他测试(PRSV-HN,CaM,和Chy与ABI7500和QS12K,使用LC480)进行的所有检测测试均显示,使用两种DNA聚合酶进行的每次测试的LOD均无重要差异。因此,用ABI7500或QS12K进行PRSV-YK和PRSV-SC检测测试时,FastGene应用于避免在低混合水平下含有GM木瓜系PRSV-YK和PRSV-SC的食物的假阴性。
    In the Japanese official detection method for unauthorized genetically modified (GM) papayas, one of two types of real-time PCR reagents with DNA polymerase (TaqMan Gene Master Mix [TaqMan Gene] or FastGene QPCR Probe Mastermix w/ROX [FastGene]) is primarily used for measurement. In 2022, we conducted a laboratory performance study on the unauthorized GM papaya line PRSV-YK, and the results revealed that high threshold cycle (Cq) values for the PRSV-YK detection test were obtained using TaqMan Gene with the 7500 Fast & 7500 Real-Time PCR System (ABI7500) and QuantStudio 12K Flex (QS12K), indicating the possibility of false negatives. The possibility of similar problems with all unauthorized GM papaya lines detection tests needs to be evaluated. In this study, we performed detection tests on unauthorized GM papaya lines (PRSV-YK, PRSV-SC, and PRSV-HN), the cauliflower mosaic virus 35S promotor (CaM), and a papaya positive control (Chy), and examined how the limits of detection (LOD) for each test are affected by two types of DNA polymerases (TaqMan Gene and FastGene) and three types of real-time PCR instruments (ABI7500, QS12K, and LightCycler 480 Instrument II [LC480]). In the PRSV-YK and PRSV-SC detection tests using ABI7500 and QS12K, measurement with TaqMan Gene showed a higher LOD than FastGene. In this case, an exponential amplification curve was confirmed on the amplification plot; however, the amplification curve did not cross the ΔRn threshold line and the correct Cq value was not obtained with a threshold line=0.2. The other tests (PRSV-HN, CaM, and Chy with ABI7500 and QS12K, and all detection tests with LC480) showed no important differences in the LOD for each test using either DNA polymerase. Therefore, when performing PRSV-YK and PRSV-SC detection tests with the ABI7500 or QS12K, FastGene should be used to avoid false negatives for foods containing GM papaya lines PRSV-YK and PRSV-SC at low mixing levels.
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  • 文章类型: Journal Article
    包括水稻在内的20多种转基因粮食作物已在许多国家获得批准。转基因水稻及其衍生产品尚未在印度获得批准,因此在印度被视为未经授权的转基因生物。因此,重要的是要跟踪是否含有大米食品,市场上可用的是无转基因的。
    进行了一项试点研究,以检查30个包装米粒和以大米为原料的加工食品样品的转基因状态,使用针对花椰菜花叶病毒35S启动子(P-35S)的聚合酶链反应(PCR)测定法,茉莉碱合成酶终止子(T-nos),膦丝菌素-N-乙酰转移酶(pat)和cry1Ac基因,这可以涵盖所有全球批准的转基因水稻事件的筛查。
    根据结果,测试的样本均未发现P-35S阳性,T-nos,pat和cry1Ac。
    在测试的样品中未检测到未经授权的转基因大米成分。可以进一步进行此类研究,以测试从各个谷类作物的转基因事件获得批准的国家进口的食品中除大米以外的谷物的转基因成分。作为监管要求的一部分。
    UNASSIGNED: More than 20 genetically modified (GM) food crops including rice have been approved in many countries. GM rice and derived products have not yet been approved in India so they are considered as unauthorized genetically modified organisms (GMOs) in the country. Therefore it is important to track whether the rice containing food items, available in the marketplace are GMO-free.
    UNASSIGNED: A pilot study was conducted to check the GM status of 30 samples of packed rice grains and processed food products with rice as an ingredient, using polymerase chain reaction (PCR) assays targeting Cauliflower Mosaic Virus 35 S promoter (P-35 S), nopaline synthase terminator (T-nos), phosphinothricin-N-acetyltransferase (pat) and cry1Ac gene, which could cover screening for all the globally approved GM rice events.
    UNASSIGNED: Based on the results, none of the samples tested were found positive for P-35 S, T-nos, pat and cry1Ac.
    UNASSIGNED: The unauthorized presence of GM rice ingredients was not detected in the samples tested. Such studies may further be conducted for the testing of GM ingredients derived from cereals other than rice in the food products imported from the country where GM events of respective cereal crop are approved, as a part of regulatory requirement.
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  • 文章类型: Journal Article
    大肠杆菌O157:H7,金黄色葡萄球菌,沙门氏菌和沙门氏菌是主要的食源性病原体,在自然界中普遍存在,并负责几次食品安全事故的爆发。因此,开发了一种快速实用的技术(PMA-mPCR),用于同时检测活的大肠杆菌O157:H7,金黄色葡萄球菌,纯培养和食物基质中的沙门氏菌。为了消除假阳性结果,应用单叠氮化物丙啶(PMA)来选择性地抑制死细胞的DNA扩增。结果表明,PMA的最佳浓度为5.0μg/mL。在培养液中,mPCR的检测限为103CFU/mL,通过PMA-mPCR,在纯培养物和食品基质(牛奶和碎牛肉)中均为104CFU/mL。此外,本研究还探索了混合活细胞和死细胞的检测。活菌计数和死亡计数的检测灵敏度比小于1:10。因此,这里提出的PMA-mPCR测定法可能为同时检测活的大肠杆菌O157:H7,金黄色葡萄球菌,和沙门氏菌,对VBNC细胞的检测和浓度评价也有很大的潜力。
    Escherichia coli O157:H7, Staphylococcus aureus, and Salmonella are major foodborne pathogens that are widespread in nature and responsible for several outbreaks of food safety accidents. Thus, a rapid and practical technique (PMA-mPCR) was developed for the simultaneous detection of viable E. coli O157:H7, S. aureus, and Salmonella in pure culture and in a food matrix. To eliminate false positive results, propidium monoazide (PMA) was applied to selectively suppress the DNA amplification of dead cells. The results showed the optimum concentration of PMA is 5.0 µg/mL. The detection limit of this assay by mPCR was 103 CFU/mL in the culture broth, and by PMA-mPCR was 104 CFU/mL both in pure culture and a food matrix (milk and ground beef). In addition, the detection of mixed viable and dead cells was also explored in this study. The detection sensitivity ratio of viable and dead counts was less than 1:10. Therefore, the PMA-mPCR assay proposed here might provide an efficient detection tool for the simultaneous detection of viable E. coli O157:H7, S. aureus, and Salmonella and also have great potential for the detection and concentration assessment of VBNC cells.
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  • 文章类型: Journal Article
    杂交育种是香菇育种中最常用的方法,然而,杂种的低结实率一直给育种者带来麻烦和障碍。结果能力的早期筛选方法可以使育种工作更有效。在本文中,建立了一种基于琼脂扩散原理的快速高通量漆酶活性检测方法。这样,我们研究了在不同培养基上的L.edodes育种群体中36个菌株的组成型和诱导型细胞外漆酶活性,并对这些菌株的结果进行了相关分析。结果表明,在非诱导培养基中培养8d的菌丝体的漆酶活性可作为早期筛选指标,以判断其后期是否具有结果能力。基于菌丝体漆酶活性特征,建立了早期快速简便的杂种群体筛选方法。使用来自另外5个不同杂种群体的127个菌株来验证早期筛选方法。从验证结果来看,早期筛查方法是有效的,但是需要适当的筛选阈值来根据交叉人群进行选择,这将大大提高香菇的育种效率。
    Crossbreeding is the most commonly used method in breeding of Lentinula edodes, however low fruiting rate of the hybrids has always caused troubles and barriers for breeders. An early screening method of the fruiting ability could make the breeding work more efficient. In this paper, a rapid and high-throughput laccase activity detection method based on agar diffusion principle was developed. In this way, we investigated the constitutive and inducible extracellular laccase activity of 36 strains in a breeding population of L. edodes on different media and performed a correlation analysis with fruiting ability of these strains. The results showed the laccase activity of mycelium cultured in non-induced medium for 8 d could be used as an early screening index to judge whether it had fruiting ability at the later stage. Early rapid and simple screening method for hybrid populations was established based on laccase activity characteristics of mycelia. 127 strains from another 5 different hybrid populations were used to verify the early screening method. From the validation results, the early screening method was effective, but the appropriate screening threshold was needed to select according to the cross population, which would greatly to improve the breeding efficiency of L. edodes.
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  • 文章类型: Journal Article
    地外生命的探测和分析是空间科学的重要课题。火星是探索外星生命的最重要的行星之一,由于它的物理特性和它的古老和现在的环境,揭示了以前的探索任务。在本文中,我们对探测外星生命和与生命有关的物质的方法进行了比较研究。为此,我们对太阳系探测任务中外星生命探测的目标特征及其评估方法进行了分类和总结。提供了一个汇总表。我们得出的结论是,在这一刻(i)没有现实的单一检测方法能够结束外星生命的发现,(ii)没有一种方法在所有方面都优于其他方法,(iii)没有单一的方法能够区分外星生命和陆地生命。因此,互补方法的组合是必不可少的。我们强调努力探测外星生命而不忽视可能的外星生命形式的重要性,甚至以容忍假阳性为代价。目标和检测方法的总结应不断更新,应该进行两者的比较研究。尽管这项研究假设火星是生命搜索的主要环境的模型站点,本文描述的目标和检测方法对于在任何天体环境中搜索外星生命以及对返回样本的初始检查也是有用的。
    The detection and analysis of extraterrestrial life are important issues of space science. Mars is among the most important planets to explore for extraterrestrial life, owing both to its physical properties and to its ancient and present environments as revealed by previous exploration missions. In this paper, we present a comparative study of methods for detecting extraterrestrial life and life-related substances. To this end, we have classified and summarized the characteristics targeted for the detection of extraterrestrial life in solar system exploration mission and the methods used to evaluate them. A summary table is presented. We conclude that at this moment (i) there is no realistic single detection method capable of concluding the discovery of extraterrestrial life, (ii) no single method has an advantage over the others in all respects, and (iii) there is no single method capable of distinguishing extraterrestrial life from terrestrial life. Therefore, a combination of complementary methods is essential. We emphasize the importance of endeavoring to detect extraterrestrial life without overlooking possible alien life forms, even at the cost of tolerating false positives. Summaries of both the targets and the detection methods should be updated continuously, and comparative studies of both should be pursued. Although this study assumes Mars to be a model site for the primary environment for life searches, both the targets and detection methods described herein will also be useful for searching for extraterrestrial life in any celestial environment and for the initial inspection of returned samples.
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  • 文章类型: Journal Article
    An accurate and simple HPLC-UV method has been developed for the determination of clonidine in mouse plasma. A reversed phase C18 Nova Pack® column (125 mm × 4.6 mm i.d., × 3 μm particle size) was used as stationary phase. The mobile phase composition was a mixture of 0.1% diethylamine/acetonitrile (70:30, v/v) at pH 8 in an isocratic mode at flow rate was 1.0 mL/min. Detection was set at 210 nm. Tizanidine was used as an internal standard. The clonidine and tizanidine were extracted from plasma matrix using the deproteinization technique. The developed method exhibited a linear calibration range 100.0-2000 ng/mL and the lower limit of detection (LOD) and quantification (LOQ) were 31.0 and 91.9 ng/mL, respectively. The intra-day and inter-day accuracy and precision of the method were within 8.0% and 3.0%, respectively, relative to the nominal concentration. The developed method was validated with respect to linearity, accuracy, precision, and selectivity according to the US Food and drug guideline. Minimal degradation was demonstrated during the determination of clonidine under different stability conditions. The suggested method has been successfully applied during a pharmacokinetic study of clonidine in mouse plasma.
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  • 文章类型: Journal Article
    在细胞/器官发育和发病机制中,基因组组织的多个维度在基因表达的非编码区调控中起着至关重要的作用。多维基因组结构的精确测量可确保数据质量,并完全取决于研究设计。我们在这里概述了基因组结构检测和分析中使用的方法的数量,并比较了基于3C的方法的优缺点,基于PCR扩增,和基于测序的测量。我们讨论了根据靶向基因组位点优化各种技术,所需的决议,和可能的技术偏见。评估了不同分析工具和基于计算系统的自动分析的比较,以定义3D基因组研究中数据分析的更多机会和挑战。基因组结构在通过增强子-启动子相互作用组织的单个基因组的水平上可视化,TAD,以及TAD之间的染色体内和染色体间相互作用。因此,多维基因组组织的方法不仅在3D基因组调控转录的研究中至关重要,而且还发现了疾病特异性生物标志物和用于诊断和治疗的靶标。
    Multiple dimensions of genome organization play critical roles in the regulation of non-coding regions in gene expression in cell/organ development and pathogenesis. Precise measurements of multi-dimensional genome structure ensure data quality and fully depend upon the study design. We here overview the number of methodologies used in the detection and analysis of genome structure and compare advantages and disadvantages of 3C-based, PCR amplification-based, and sequencing-based measurements. We discuss about the optimization of various techniques according to targeted genomic sites, the required resolution, and possible technique biases. Comparison of different analysis tools and computational system-based automatic analysis is evaluated to define more opportunities and challenges of data analysis in 3D genome research. The genome structure is visualized in levels of single genome organized by enhancer-promoter interactions, TAD, and intra-chromosomal and inter-chromosomal interactions between TADs. Thus, methodologies of genome organization multi-dimensions are not only critical in studies on 3D genome-regulated transcriptions, but also in the discovery of disease-specific biomarkers and targets for diagnosis and therapies.
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  • 文章类型: Clinical Trial
    Recently, the prognostic significance of circulating tumor cells (CTCs) in primary breast cancer as assessed using the Food-and-Drug-Administration-approved CellSearch® system has been demonstrated. Here, we evaluated the prognostic relevance of CTCs, as determined using manually performed immunocytochemistry (MICC) in peripheral blood at primary diagnosis, in patients from the prospectively randomized multicenter SUCCESS-A trial (EudraCT2005000490-21).
    We analyzed 23 ml of blood from 1221 patients with node-positive or high risk node-negative breast cancer before adjuvant taxane-based chemotherapy. Cells were separated using a density gradient followed by epithelial cell labeling with the anti-cytokeratin-antibody A45-B/B3, immunohistochemical staining with new fuchsin, and cytospin preparation. All cytospins were screened for CTCs, and the cutoff for positivity was at least one CTC. The prognostic value of CTCs with regard to disease-free survival (DFS), distant disease-free survival (DDFS), breast-cancer-specific survival (BCSS), and overall survival (OS) was assessed using both univariate analyses applying the Kaplan-Meier method and log-rank tests, and using multivariate Cox regressions adjusted for other predictive factors.
    In 20.6 % of all patients (n = 251) a median of 1 (range, 1-256) CTC was detected, while 79.4 % of the patients (n = 970) were negative for CTCs before adjuvant chemotherapy. A pT1 tumor was present in 40.0 % of patients, 4.8 % had G1 grading and 34.6 % were node-negative. There was no association between CTC positivity and tumor stage, nodal status, grading, histological type, hormone receptor status, Her2 status, menopausal status or treatment. Univariate survival analyses based on a median follow-up of 64 months revealed no significant differences between CTC-positive and CTC-negative patients with regard to DFS, DDFS, BCSS, or OS. This was confirmed by fully adjusted multivariate Cox regressions, showing that the presence of CTCs (yes/no) as assessed by MICC did not predict DFS, DDFS, BCSS or OS.
    We could not demonstrate prognostic relevance regarding CTCs that were quantified using the MICC method at the time of primary diagnosis in our cohort of early breast cancer patients. Further studies are necessary to evaluate if the presence of CTCs assessed using MICC has prognostic relevance, or can be used for risk stratification and treatment monitoring in adjuvant breast cancer.
    The ClinicalTrial.gov registration ID of this prospectively randomized trial is NCT02181101 ; the (retrospective) registration date was June 2014 (study start date September 2005).
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