detection method

检测方法
  • 文章类型: Journal Article
    猪deltacoronavirus(PDCoV)是一种新发现的猪肠道致病性冠状病毒,具有单链正义RNA基因组和包膜。PDCoV感染不同年龄的猪,并导致新生仔猪急性腹泻和呕吐。在严重的情况下,感染导致脱水,疲惫,和病态仔猪的死亡,给养猪场带来巨大的经济损失。PDCoV感染的临床症状与其他猪肠道病毒非常相似。尽管不进行测试很难区分这些病毒感染,监测PDCoV非常重要,因为它可以在人群中传播。最常用的检测PDCoV的方法是qPCR,这是耗时的,需要熟练的人员和设备。许多农场无法满足检测所需的条件。因此,有必要建立一种更快速、更方便的检测PDCoV的方法。
    通过将RPA(重组酶聚合酶等温扩增)与CRISPR/Cas13a相结合,建立一种快速便捷的PDCoV检测方法。
    设计了PDCoV的特异性RPA引物和crRNA,样品中的核酸用RPA扩增。进行荧光CRISPR/Cas13a检测。我们使用qPCR作为对照方法评估了RPA-CRISPR/Cas13a测定的灵敏度和特异性。
    CRISPR/Cas13a辅助检测在90分钟内完成。PDCoV的最低检测限为5.7×101拷贝/μL。特异性分析显示该测定不与其它三种猪肠道病毒发生交叉反应。
    RPA-CRISPR/Cas13a方法具有灵敏度高的优点,特异性强,快速反应,和容易获得的结果,并可用于PDCoV的检测。
    UNASSIGNED: Porcine deltacoronavirus (PDCoV) is a newly discovered porcine intestinal pathogenic coronavirus with a single-stranded positive-sense RNA genome and an envelope. PDCoV infects pigs of different ages and causes acute diarrhea and vomiting in newborn piglets. In severe cases, infection leads to dehydration, exhaustion, and death in sick piglets, entailing great economic losses on pig farms. The clinical symptoms of PDCoV infection are very similar to those of other porcine enteroviruses. Although it is difficult to distinguish these viral infections without testing, monitoring PDCoV is very important because it can spread in populations. The most commonly used methods for the detection of PDCoV is qPCR, which is time-consuming and require skilled personnel and equipment. Many farms cannot meet the conditions required for detection. Therefore, it is necessary to establish a faster and more convenient method for detecting PDCoV.
    UNASSIGNED: To establish a rapid and convenient detection method for PDCoV by combining RPA (Recombinase Polymerase Isothermal Amplification) with CRISPR/Cas13a.
    UNASSIGNED: Specific RPA primers and crRNA for PDCoV were designed, and the nucleic acids in the samples were amplified with RPA. Fluorescent CRISPR/Cas13a detection was performed. We evaluated the sensitivity and specificity of the RPA-CRISPR/Cas13a assay using qPCR as the control method.
    UNASSIGNED: CRISPR/Cas13a-assisted detection was completed within 90 min. The minimum detection limit of PDCoV was 5.7 × 101 copies/μL. A specificity analysis showed that the assay did not cross-react with three other porcine enteroviruses.
    UNASSIGNED: The RPA-CRISPR/Cas13a method has the advantages of high sensitivity, strong specificity, fast response, and readily accessible results, and can be used for the detection of PDCoV.
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  • 文章类型: Journal Article
    众多具有优异的光捕获和光热转换能力的纳米材料已广泛应用于各个领域,如光热诊断和治疗,微量物质检测,和光学成像。尽管近年来已经建立了利用纳米材料的光热效应的光热检测方法,关于它们在食品安全检测中的应用的评论很少。在这里,光热转换机制的最新进展,光热转换效率计算,综述了光热纳米材料的制备方法。特别是,全面论述了光热纳米材料在各种食品危害分析中的应用以及新建立的光热检测方法。此外,讨论了基于光热纳米材料的检测方法的发展和未来的发展趋势,这为研究者提出更有效的,敏感,和准确的检测方法。
    Numerous nanomaterials endowed with outstanding light harvesting and photothermal conversion abilities have been extensively applied in various fields, such as photothermal diagnosis and therapy, trace substance detection, and optical imaging. Although photothermal detection methods have been established utilizing the photothermal effect of nanomaterials in recent years, there is a scarcity of reviews regarding their application in food safety detection. Herein, the recent advancements in the photothermal conversion mechanism, photothermal conversion efficiency calculation, and preparation method of photothermal nanomaterials were reviewed. In particular, the application of photothermal nanomaterials in various food hazard analyses and the newly established photothermal detection methods were comprehensively discussed. Moreover, the development and promising future trends of photothermal nanomaterial-based detection methods were discussed, which provide a reference for researchers to propose more effective, sensitive, and accurate detection methods.
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  • 文章类型: Journal Article
    蔬菜病害的自动检测和识别可以提高蔬菜品质,增加利润。温室种植的蔬菜疾病的图像通常具有复杂的背景,各种各样的疾病,和微妙的症状差异。以前的研究已经努力准确地确定病变位置和量化感染程度,导致整体识别率较低。为了应对验证数据集不足以及检测和识别率低带来的挑战,本研究利用寿光的地理优势,被誉为“蔬菜镇”,\"建立自建蔬菜基地进行数据采集和验证实验。集中于患有各种疾病的广泛的水果和蔬菜作物,我们进行了温室病害图像的现场采集,编制了一个大规模的数据集,并介绍了时空融合注意力网络(STFAN)。STFAN整合了蔬菜病害发生的多源信息,增强模型的弹性。此外,我们提出了多层编码器-解码器特征融合网络(MEDFFN)来抵消深度卷积块中的特征消失,辅以边界结构损失函数,以指导模型获取更详细,更准确的边界信息。通过设计从多个源提取高分辨率特征表示的检测和识别模型,实现了疾病的精确检测和识别。本研究为蔬菜病害的整体防控提供了技术支持,从而推进智慧农业。结果表明,在我们自建的VDGE数据集上,与YOLOv7-tiny相比,YOLOv8n,和YOLOv9,提出的模型(多源信息融合方法,用于蔬菜病害检测,MIFV)使MAP提高了3.43%,3.02%,和2.15%,分别,展示显著的性能优势。MIFV模型参数为39.07M,计算复杂度为108.92GFLOPS,突出突出的实时性和检测精度相比主流算法。这项研究表明,提出的MIFV模型可以以更低的成本快速准确地检测和识别温室环境中的蔬菜疾病。
    Automated detection and identification of vegetable diseases can enhance vegetable quality and increase profits. Images of greenhouse-grown vegetable diseases often feature complex backgrounds, a diverse array of diseases, and subtle symptomatic differences. Previous studies have grappled with accurately pinpointing lesion positions and quantifying infection degrees, resulting in overall low recognition rates. To tackle the challenges posed by insufficient validation datasets and low detection and recognition rates, this study capitalizes on the geographical advantage of Shouguang, renowned as the \"Vegetable Town,\" to establish a self-built vegetable base for data collection and validation experiments. Concentrating on a broad spectrum of fruit and vegetable crops afflicted with various diseases, we conducted on-site collection of greenhouse disease images, compiled a large-scale dataset, and introduced the Space-Time Fusion Attention Network (STFAN). STFAN integrates multi-source information on vegetable disease occurrences, bolstering the model\'s resilience. Additionally, we proposed the Multilayer Encoder-Decoder Feature Fusion Network (MEDFFN) to counteract feature disappearance in deep convolutional blocks, complemented by the Boundary Structure Loss function to guide the model in acquiring more detailed and accurate boundary information. By devising a detection and recognition model that extracts high-resolution feature representations from multiple sources, precise disease detection and identification were achieved. This study offers technical backing for the holistic prevention and control of vegetable diseases, thereby advancing smart agriculture. Results indicate that, on our self-built VDGE dataset, compared to YOLOv7-tiny, YOLOv8n, and YOLOv9, the proposed model (Multisource Information Fusion Method for Vegetable Disease Detection, MIFV) has improved mAP by 3.43%, 3.02%, and 2.15%, respectively, showcasing significant performance advantages. The MIFV model parameters stand at 39.07 M, with a computational complexity of 108.92 GFLOPS, highlighting outstanding real-time performance and detection accuracy compared to mainstream algorithms. This research suggests that the proposed MIFV model can swiftly and accurately detect and identify vegetable diseases in greenhouse environments at a reduced cost.
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  • 文章类型: Journal Article
    解决当前铁路轨道异物检测技术的局限性,其缺点是实时性能不足和检测小物体的准确性降低,本文介绍了一种创新的基于视觉的感知方法,利用深度学习的力量。这种方法的核心是利用复杂的轨道检测方法构建铁路边界模型,以及增强的UNet语义分割网络,以实现对不同轨道类别的自主分割。通过采用等间隔除法和逐行遍历,精确提取临界航迹特征点,通过最小二乘法推导出轨道线性方程,从而建立了准确的铁路边界模型。我们从四个方面对YOLOv5s检测模型进行了优化:将SE注意机制纳入颈部网络层,以增强模型的特征提取能力,添加预测层以提高小物体的检测性能,提出了一种线性尺寸缩放方法来获得合适的锚盒,利用Inner-IoU细化边界回归损失函数,从而提高边界框的定位精度。我们使用自构建的图像数据集对铁路轨道异物入侵进行了检测准确性验证。结果表明,所提出的语义分割模型实现了91.8%的MIoU,比以前的模型提高了3.9%,有效地分割铁路轨道。此外,优化后的检测模型可以有效地检测异物对轨道的侵入,与原始YOLOv5s模型相比,减少了漏报和误报,平均精度提高了7.4%(IoU=0.5)。该模型在涉及小对象的场景中表现出强大的泛化能力。该方法对铁路轨道异物入侵检测的深度学习技术进行了有效探索,适合在复杂环境中使用,确保铁路线的运行安全。
    Addressing the limitations of current railway track foreign object detection techniques, which suffer from inadequate real-time performance and diminished accuracy in detecting small objects, this paper introduces an innovative vision-based perception methodology harnessing the power of deep learning. Central to this approach is the construction of a railway boundary model utilizing a sophisticated track detection method, along with an enhanced UNet semantic segmentation network to achieve autonomous segmentation of diverse track categories. By employing equal interval division and row-by-row traversal, critical track feature points are precisely extracted, and the track linear equation is derived through the least squares method, thus establishing an accurate railway boundary model. We optimized the YOLOv5s detection model in four aspects: incorporating the SE attention mechanism into the Neck network layer to enhance the model\'s feature extraction capabilities, adding a prediction layer to improve the detection performance for small objects, proposing a linear size scaling method to obtain suitable anchor boxes, and utilizing Inner-IoU to refine the boundary regression loss function, thereby increasing the positioning accuracy of the bounding boxes. We conducted a detection accuracy validation for railway track foreign object intrusion using a self-constructed image dataset. The results indicate that the proposed semantic segmentation model achieved an MIoU of 91.8%, representing a 3.9% improvement over the previous model, effectively segmenting railway tracks. Additionally, the optimized detection model could effectively detect foreign object intrusions on the tracks, reducing missed and false alarms and achieving a 7.4% increase in the mean average precision (IoU = 0.5) compared to the original YOLOv5s model. The model exhibits strong generalization capabilities in scenarios involving small objects. This proposed approach represents an effective exploration of deep learning techniques for railway track foreign object intrusion detection, suitable for use in complex environments to ensure the operational safety of rail lines.
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  • 文章类型: Journal Article
    使用针对大肠杆菌的传统方法和当前分子技术的大肠杆菌细菌的检测和计数通常涉及具有较低灵敏度和特异性的长过程,以区分用于微生物水分析的活细菌和非活细菌。该方法涉及开发和验证包括由微通道网络包围的特定抗体官能化的环形共振器的免疫传感器,作为用于检测和间接计数用于消耗的水样品中的大肠杆菌的替代方法。进行不同的ELISA测定以表征选择作为大肠杆菌的特异性B-半乳糖苷酶和膜LPS抗原的检测探针的单克隆和多克隆抗体。在免疫传感器中使用的氮化硅表面上进行了固定对照研究,用从ELISA测定中选择的抗体固定。该方法的特异性通过在将各种消毒方法应用于旨在供人类消费的水样品后,从活的和非活的目标细菌中检测少至10CFU/mL的大肠杆菌来确认。通过对免疫传感器耦合微流体系统的综合评估,验证了该方法的100%检测率和100CFU/mL定量限。涉及至少50个浓度范围为10至106CFU/mL的目标细菌和50个经过和未经消毒处理污染的实际样品。从结果获得的每个校准曲线计算得出的相关系数约为1,表明具有灵敏和快速的检测能力,适用于食品工业中人类消费的水资源。生物传感器显示在不到4小时内提供结果,允许快速识别微生物污染对于确保与食品安全或环境诊断相关的水监测至关重要,并允许及时干预以减轻污染风险。的确,所实现的设置促进了实验室过程的现场执行,允许检测有活力和无活力的细菌,这意味着在同一污染样品中同时检测病原体的未来发展。
    Detection and enumeration of coliform bacteria using traditional methods and current molecular techniques against E. coli usually involve long processes with less sensitivity and specificity to distinguish between viable and non-viable bacteria for microbiological water analysis. This approach involves developing and validating an immunosensor comprising ring resonators functionalized with specific antibodies surrounded by a network of microchannels as an alternative method for detecting and indirectly enumerating Escherichia coli in samples of water for consumption. Different ELISA assays were conducted to characterize monoclonal and polyclonal antibodies selected as detection probes for specific B-galactosidase enzymes and membrane LPS antigens of E. coli. An immobilization control study was performed on silicon nitride surfaces used in the immunosensor, immobilized with the selected antibodies from the ELISA assays. The specificity of this method was confirmed by detecting as few as 10 CFU/mL of E. coli from viable and non-viable target bacteria after applying various disinfection methods to water samples intended for human consumption. The 100% detection rate and a 100 CFU/mL Limit of Quantification of the proposed method were validated through a comprehensive assessment of the immunosensor-coupled microfluidic system, involving at least 50 replicates with a concentration range of 10 to 106 CFU/mL of the target bacteria and 50 real samples contaminated with and without disinfection treatment. The correlation coefficient of around one calculated for each calibration curve obtained from the results demonstrated sensitive and rapid detection capabilities suitable for application in water resources intended for human consumption within the food industry. The biosensor was shown to provide results in less than 4 h, allowing for rapid identification of microbial contamination crucial for ensuring water monitoring related to food safety or environmental diagnosis and allowing for timely interventions to mitigate contamination risks. Indeed, the achieved setup facilitates the in situ execution of laboratory processes, allowing for the detection of both viable and non-viable bacteria, and it implies future developments of simultaneous detection of pathogens in the same contaminated sample.
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  • 文章类型: Journal Article
    RT-qPCR允许检测病毒和监测病毒复制。这项技术在SARS-CoV-2大流行期间被广泛使用,在那里它证明了它的效率和鲁棒性。在这里,我们描述了随着时间的推移,裂谷热和托斯卡纳病毒感染的分析,通过RT-qPCR定量病毒基因组。我们进一步阐述了通过在逆转录步骤中使用对每条链特异的引物来区分基因组和反基因组病毒RNA的方法。
    RT-qPCR allows the detection of viruses and the monitoring of viral replication. This technique was extensively employed during the SARS-CoV-2 pandemic, where it demonstrated its efficiency and robustness. Here we describe the analysis of Rift Valley fever and Toscana virus infections over time, achieved through the RT-qPCR quantification of the viral genome. We further elaborate on the method to discriminate between genomic and antigenomic viral RNAs by using primers specific for each strand during the reverse transcription step.
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  • 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
    需要对多种食物过敏原进行快速分析,以确认加工食品中食物过敏原标签的适当性。本研究旨在开发一种快速可靠的方法,同时检测痕量的七种食品致敏性蛋白(小麦,荞麦,牛奶,鸡蛋,甲壳类动物,花生,和核桃)在加工食品中使用LC-MS/MS。使用悬浮捕集(S-Trap)柱和在线自动固相萃取来改善以前使用LC-MS/MS进行过敏原分析所需的复杂且耗时的预处理过程。所开发的方法能够同时检测来自五种类型的经痕量变应原性蛋白质修正的事件样品中的七种食品成分的特定蛋白质的选定标记肽。估计每种蛋白质的方法的检测值的极限 Rapid analysis of multiple food allergens is required to confirm the appropriateness of food allergen labelling in processed foods. This study aimed to develop a rapid and reliable method to simultaneously detect trace amounts of seven food allergenic proteins (wheat, buckwheat, milk, egg, crustacean, peanut, and walnut) in processed foods using LC-MS/MS. Suspension-trapping (S-Trap) columns and on-line automated solid-phase extraction were used to improve the complex and time-consuming pretreatment process previously required for allergen analysis using LC-MS/MS. The developed method enabled the simultaneous detection of selected marker peptides for specific proteins derived from seven food ingredients in five types of incurred samples amended with trace amounts of allergenic proteins. The limit of detection values of the method for each protein were estimated to be <1 mg/kg. The developed analytical approach is considered an effective screening method for confirming food allergen labelling on a wide range of processed foods.
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  • 文章类型: Journal Article
    一种新的创新方法,MICA军团菌,允许在2天内自动计数生活水样中的嗜肺军团菌,每个测试部分的检测限为2CFU。在这里,我们表明它在7到15天内给出了与法国标准方法NFT90-431获得的结果相同的结果。
    A new innovative method, MICA Legionella, allows for the automatic enumeration of Legionella pneumophila in domestic water samples in 2 days, with a detection limit of 2 CFU per test portion. Here we show that it gives equivalent results to those obtained by the French standard method NF T90-431 in 7 to 15 days.
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