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Optimizing Western blotting immunodetection: Streamlining antibody cocktails for reduced protocol time and enhanced multiplexing applications. 优化 Western 印迹免疫检测:精简鸡尾酒抗体,缩短方案时间,增强多重应用。
IF 2.5 Q3 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-10-15 eCollection Date: 2024-01-01 DOI: 10.1093/biomethods/bpae077
L Z Yamani, Khaldoon Alsamman, Omar S El-Masry

Adaptive, rather than innate, immunity relies mainly on antigen-antibody recognition. This recognition is driven by the binding of specific antibody paratopes to distinct epitopes found on antigens. This interaction is pivotal for immune responses that have been re-purposed for diagnostic and therapeutic purposes. This article focuses on Western blotting, an in vitro technique performed for protein immunodetection. Traditionally, this technique requires separate incubations of both primary and secondary antibodies, for which these antibodies recognize different antigen epitopes (conventional method). We propose a modified protocol combining both antibodies, involving a single incubation step that reduces time and conserves reagents (non-conventional/improved method). This improved protocol will enhance efficiency without compromising detection accuracy. It will support multiplexing, enabling the simultaneous detection of multiple proteins. Despite the positive results found by applying available antibodies, further optimization is required for a more thorough evaluation, to ensure that all antibodies consistently yield successful results in every detection attempt for broader use. Our findings indicate that the tested antibody cocktails remained stable over time, which suggests potential for commercialization of this modified Western blot protocol with a wide scope towards multiplex diagnostic application.

适应性免疫而非先天性免疫主要依靠抗原-抗体识别。这种识别是由特异性抗体副位点与抗原上的不同表位结合驱动的。这种相互作用是免疫反应的关键,已被重新用于诊断和治疗目的。本文重点介绍 Western 印迹技术,这是一种用于蛋白质免疫检测的体外技术。传统上,这种技术需要一抗和二抗分别孵育,而这些抗体能识别不同的抗原表位(传统方法)。我们提出了一种结合两种抗体的改进方案,只需一个孵育步骤,既缩短了时间,又节省了试剂(非常规/改进方法)。这一改进方案将在不影响检测准确性的前提下提高效率。它还支持多重检测,可同时检测多种蛋白质。尽管应用现有抗体取得了积极的结果,但还需要进一步优化,进行更全面的评估,以确保所有抗体在每次检测中都能取得一致的成功结果,从而得到更广泛的应用。我们的研究结果表明,经过测试的抗体鸡尾酒在一段时间内保持稳定,这表明这种改良的 Western 印迹方案具有商业化的潜力,可广泛应用于多重诊断。
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引用次数: 0
Live cell fluorescence microscopy-an end-to-end workflow for high-throughput image and data analysis. 活细胞荧光显微镜--用于高通量图像和数据分析的端到端工作流程。
IF 2.5 Q3 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-10-11 eCollection Date: 2024-01-01 DOI: 10.1093/biomethods/bpae075
Jakub Zahumensky, Jan Malinsky

Fluorescence microscopy images of biological samples contain valuable information but require rigorous analysis for accurate and reliable determination of changes in protein localization, fluorescence intensity, and morphology of the studied objects. Traditionally, cells for microscopy are immobilized using chemicals, which can introduce stress. Analysis often focuses only on colocalization and involves manual segmentation and measurement, which are time-consuming and can introduce bias. Our new workflow addresses these issues by gently immobilizing cells using a small agarose block on a microscope coverslip. This approach is suitable for cell-walled cells (yeast, fungi, plants, bacteria), facilitates their live imaging under conditions close to their natural environment and enables the addition of chemicals during time-lapse experiments. The primary focus of the protocol is on the presented analysis workflow, which is applicable to virtually any cell type-we describe cell segmentation using the Cellpose software followed by automated analysis of a multitude of parameters using custom-written Fiji (ImageJ) macros. The results can be easily processed using the provided R markdown scripts or available graphing software. Our method facilitates unbiased batch analysis of large datasets, improving the efficiency and accuracy of fluorescence microscopy research. The reported sample preparation protocol and Fiji macros were used in our recent publications: Microbiol Spectr (2022), DOI: 10.1128/spectrum.01961-22; Microbiol Spectr (2022), DOI: 10.1128/spectrum.02489-22; J Cell Sci (2023), DOI: 10.1242/jcs.260554.

生物样本的荧光显微图像包含宝贵的信息,但需要进行严格的分析,才能准确可靠地确定所研究对象的蛋白质定位、荧光强度和形态的变化。传统上,用于显微镜观察的细胞是用化学物质固定的,这会带来应力。分析通常只关注共定位,涉及手动分割和测量,既费时又可能产生偏差。我们的新工作流程通过在显微镜盖玻片上使用小型琼脂糖块轻轻固定细胞来解决这些问题。这种方法适用于细胞壁细胞(酵母、真菌、植物、细菌),便于在接近其自然环境的条件下对其进行活体成像,并能在延时实验中添加化学试剂。该方案的主要重点是所介绍的分析工作流程,它几乎适用于任何细胞类型--我们介绍了使用 Cellpose 软件进行细胞分割,然后使用定制的 Fiji(ImageJ)宏对多种参数进行自动分析。使用所提供的 R 标记脚本或可用的绘图软件可以轻松处理结果。我们的方法有助于对大型数据集进行无偏批量分析,提高荧光显微镜研究的效率和准确性。所报告的样品制备方案和 Fiji 宏被用于我们最近发表的文章中:Microbiol Spectr (2022),DOI: 10.1128/spectrum.01961-22;Microbiol Spectr (2022),DOI: 10.1128/spectrum.02489-22;J Cell Sci (2023),DOI: 10.1242/jcs.260554。
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引用次数: 0
A reproducible method to study traumatic injury-induced zebrafish brain regeneration. 研究创伤诱导斑马鱼脑再生的可重复方法。
IF 2.5 Q3 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-10-10 eCollection Date: 2024-01-01 DOI: 10.1093/biomethods/bpae073
Priyanka P Srivastava, Sidharth Bhasin, Sunita S Shankaran, Catherine Roger, Rajesh Ramachandran, Shilpi Minocha

Traumatic brain injury (TBI) can be caused by a sudden blow or jolt to the head, causing irreversible brain damage leading to cellular and functional loss. Mammals cannot repair such damage, which may increase the risk of progressive neurodegeneration. Unlike mammals, lower vertebrates such as zebrafish have the astounding capability to regenerate their brains. A model system would be of great value to study zebrafish brain regeneration. Here, we describe a physical method to induce traumatic injury in the zebrafish brain and outline a pipeline to utilize this model system to explore various aspects of brain regeneration. This will significantly advance the fields of regenerative biology and neuroscience. The method includes inducing TBI and validating this through histological assays, immunohistochemistry, and gene expression analysis. By using this model system, researchers will be able to gain valuable insights into the cellular and molecular mechanisms underlying brain regeneration. Understanding these mechanisms could lead to the identification of potential strategies to address neurodegenerative conditions in higher vertebrates.

创伤性脑损伤(TBI)可由头部突然受到撞击或颠簸引起,造成不可逆转的脑损伤,导致细胞和功能丧失。哺乳动物无法修复这种损伤,这可能会增加渐进性神经变性的风险。与哺乳动物不同,斑马鱼等低等脊椎动物具有惊人的大脑再生能力。建立一个模型系统对研究斑马鱼大脑再生具有重要价值。在此,我们描述了诱导斑马鱼大脑创伤的物理方法,并概述了利用这一模型系统探索大脑再生各方面问题的流程。这将极大地推动再生生物学和神经科学领域的发展。该方法包括诱导创伤性脑损伤,并通过组织学检测、免疫组化和基因表达分析进行验证。通过使用这一模型系统,研究人员将能够获得有关脑再生的细胞和分子机制的宝贵见解。了解了这些机制,就能找到解决高等脊椎动物神经退行性疾病的潜在策略。
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引用次数: 0
Cluster analysis identifies long COVID subtypes in Belgian patients. 聚类分析确定了比利时患者的长 COVID 亚型。
IF 2.5 Q3 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-10-09 eCollection Date: 2024-01-01 DOI: 10.1093/biomethods/bpae076
Pamela Mfouth Kemajou, Tatiana Besse-Hammer, Claire Lebouc, Yves Coppieters

Severe acute respiratory syndrome coronavirus infection presents complications known as long COVID, a multisystemic organ disease which allows multidimensional analysis. This study aims to uncover clusters of long COVID cases and establish their correlation with the clinical classification developed at the Clinical Research Unit of Brugmann University Hospital, Brussels. Such an endeavour is instrumental in customizing patient management strategies tailored to the unique needs of each distinct group. A two-stage multidimensional exploratory analysis was performed on a retrospective cohort of 205 long COVID patients, involving a factorial analysis of mixed data, and then hierarchical clustering post component analysis. The study's sample comprised 76% women, with an average age of 44.5 years. Three clinical forms were identified: long, persistent, and post-viral syndrome. Multidimensional analysis using demographic, clinical, and biological variables identified three clusters of patients. Biological data did not provide sufficient differentiation between clusters. This emphasizes the importance of identifying or classifying long COVID patients according to their predominant clinical syndrome. Long COVID phenotypes, as well as clinical forms, appear to be associated with distinct pathophysiological mechanisms or genetic predispositions. This underscores the need for further research.

严重急性呼吸系统综合征冠状病毒感染引起的并发症被称为长COVID,这是一种多系统器官疾病,可进行多维分析。本研究旨在发现长COVID病例群,并确定其与布鲁塞尔布鲁曼大学医院临床研究室制定的临床分类的相关性。这项工作有助于根据每个不同群体的独特需求定制患者管理策略。我们对 205 名长期慢性阻塞性肺病患者的回顾性队列进行了两阶段多维探索性分析,包括混合数据的因子分析和分层聚类后成分分析。研究样本中有 76% 为女性,平均年龄为 44.5 岁。确定了三种临床形式:长期、持续和病毒后综合征。利用人口统计学、临床和生物学变量进行的多维分析确定了三组患者。生物数据并不能充分区分不同群组。这强调了根据主要临床综合征对长COVID患者进行识别或分类的重要性。长 COVID 表型和临床形式似乎与不同的病理生理机制或遗传倾向有关。这凸显了进一步研究的必要性。
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引用次数: 0
Unpacking unstructured data: A pilot study on extracting insights from neuropathological reports of Parkinson's Disease patients using large language models. 解读非结构化数据:利用大型语言模型从帕金森病患者的神经病理学报告中提取见解的试点研究。
IF 2.5 Q3 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-10-04 eCollection Date: 2024-01-01 DOI: 10.1093/biomethods/bpae072
Oleg Stroganov, Amber Schedlbauer, Emily Lorenzen, Alex Kadhim, Anna Lobanova, David A Lewis, Jill R Glausier

The aim of this study was to make unstructured neuropathological data, located in the NeuroBioBank (NBB), follow Findability, Accessibility, Interoperability, and Reusability principles and investigate the potential of large language models (LLMs) in wrangling unstructured neuropathological reports. By making the currently inconsistent and disparate data findable, our overarching goal was to enhance research output and speed. The NBB catalog currently includes information from medical records, interview results, and neuropathological reports. These reports contain crucial information necessary for conducting an in-depth analysis of NBB data but have multiple formats that vary across different NBB biorepositories and change over time. In this study, we focused on a subset of 822 donors with Parkinson's disease (PD) from seven NBB biorepositories. We developed a data model with combined Brain Region and Pathological Findings data at its core. This approach made it easier to build an extraction pipeline and was flexible enough to convert resulting data to Common Data Elements, a standardized data collection tool used by the neuroscience community to improve consistency and facilitate data sharing across studies. This pilot study demonstrated the potential of LLMs in structuring unstructured neuropathological reports of PD patients available in the NBB. The pipeline enabled successful extraction of detailed tissue-level (microscopic) and gross anatomical (macroscopic) observations, along with staging information from pathology reports, with extraction quality comparable to manual curation results. To our knowledge, this is the first attempt to automatically standardize neuropathological information at this scale. The collected data have the potential to serve as a valuable resource for PD researchers, facilitating integration with clinical information and genetic data (such as genome-wide genotyping and whole-genome sequencing) available through the NBB, thereby enabling a more comprehensive understanding of the disease.

本研究旨在使神经生物库(NBB)中的非结构化神经病理学数据遵循可查找性、可访问性、互操作性和可重用性原则,并研究大型语言模型(LLM)在处理非结构化神经病理学报告方面的潜力。我们的总体目标是提高研究成果和速度,从而使目前不一致且分散的数据变得可查找。NBB 目录目前包括来自医疗记录、访谈结果和神经病理报告的信息。这些报告包含了对 NBB 数据进行深入分析所必需的关键信息,但它们有多种格式,在不同的 NBB 生物库中各不相同,而且会随着时间的推移而变化。在本研究中,我们重点研究了来自七个 NBB 生物库的 822 位帕金森病(PD)供体的子集。我们开发了一个以脑区和病理结果数据为核心的数据模型。这种方法使我们更容易建立提取管道,并能灵活地将提取的数据转换为通用数据元素(Common Data Elements),通用数据元素是神经科学界使用的标准化数据收集工具,可提高一致性并促进跨研究的数据共享。这项试点研究证明了 LLMs 在构建 NBB 中现有的帕金森病患者非结构化神经病理学报告方面的潜力。该管道成功提取了病理报告中详细的组织水平(显微镜下)和大体解剖(宏观上)观察结果以及分期信息,提取质量与人工整理结果相当。据我们所知,这是首次尝试以这种规模自动标准化神经病理学信息。收集到的数据有可能成为帕金森病研究人员的宝贵资源,促进与临床信息和通过 NBB 提供的基因数据(如全基因组基因分型和全基因组测序)的整合,从而更全面地了解该疾病。
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引用次数: 0
SMaRT M-Seq: an optimized step-by-step protocol for M protein sequencing in monoclonal gammopathies. SMaRT M-Seq:单克隆丙种球蛋白病中 M 蛋白测序的优化分步方案。
IF 2.5 Q3 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-10-03 eCollection Date: 2024-01-01 DOI: 10.1093/biomethods/bpae074
Alice Nevone, Francesca Lattarulo, Monica Russo, Pasquale Cascino, Giampaolo Merlini, Giovanni Palladini, Mario Nuvolone

In patients with monoclonal gammopathies, tumor B cells or plasma cells secrete a monoclonal antibody (M protein) that not only serves as a biomarker for tumor tracking but can also cause potentially life-threatening organ damage. This damage is driven by the patient-specific sequence of the M protein. Methods for sequencing M proteins have been limited by their high cost and time-consuming nature, and while recent approaches using next-generation sequencing or mass spectrometry offer advancements, they may require tumor cell sorting, are limited to subsets of immunoglobulin genes or patients, and/or lack extensive validation. To address these limitations, we have recently developed a novel method, termed Single Molecule Real-Time Sequencing of the M protein (SMaRT M-Seq), which combines the unbiased amplification of expressed immunoglobulin genes via inverse PCR from circularized cDNA with long-read DNA sequencing, followed by bioinformatic and immunogenetic analyses. This approach enables the unambiguous identification of full-length variable sequences of M protein genes across diverse patient cohorts, including those with low tumor burden. Our protocol has undergone technical validation and has been successfully applied to large patient cohorts with monoclonal gammopathies. Here we present the step-by-step protocol for SMaRT M-Seq. By enabling the parallel sequencing of M proteins from a large number of samples in a cost-effective and time-efficient manner, SMaRT M-Seq facilitates access to patient-specific M protein sequences, advancing personalized medicine approaches and enabling deeper mechanistic studies in monoclonal gammopathies.

在单克隆丙种球蛋白病患者中,肿瘤 B 细胞或浆细胞会分泌一种单克隆抗体(M 蛋白),这种抗体不仅是追踪肿瘤的生物标记物,还可能造成潜在的危及生命的器官损伤。这种损害是由患者特异性的 M 蛋白序列驱动的。对 M 蛋白进行测序的方法一直受限于其高昂的成本和耗时的性质,虽然最近使用下一代测序或质谱法的方法取得了进步,但它们可能需要对肿瘤细胞进行分选,局限于免疫球蛋白基因或患者的子集,和/或缺乏广泛的验证。为了解决这些局限性,我们最近开发了一种新方法,称为 M 蛋白单分子实时测序(SMaRT M-Seq),它将通过环化 cDNA 反 PCR 无偏扩增表达的免疫球蛋白基因与长线程 DNA 测序相结合,然后进行生物信息学和免疫遗传学分析。这种方法能在不同的患者队列(包括肿瘤负荷较低的患者)中明确鉴定 M 蛋白基因的全长可变序列。我们的方案已经过技术验证,并已成功应用于大型单克隆丙种球蛋白病患者队列。我们在此介绍 SMaRT M-Seq 的分步方案。SMaRT M-Seq能以低成本、高效率的方式对大量样本中的M蛋白进行平行测序,有助于获得患者特异性的M蛋白序列,从而推动个性化医疗方法的发展,并能对单克隆丙种球蛋白病进行更深入的机理研究。
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引用次数: 0
Digital approaches in post-COVID healthcare: a systematic review of technological innovations in disease management. COVID 后医疗保健中的数字化方法:疾病管理技术创新的系统回顾。
IF 2.5 Q3 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-10-01 eCollection Date: 2024-01-01 DOI: 10.1093/biomethods/bpae070
Pamela Mfouth Kemajou, Armand Mbanya, Yves Coppieters

Post-COVID conditions (PCC) emerged during the pandemic, prompting a rise in the use of Digital Health Technologies (DHTs) to manage lockdowns and hospital overcrowding. Real-time tracking and information analyses were crucial to strengthening the global research response. This study aims to map the use of modern digital approaches in estimating the prevalence, predicting, diagnosing, treating, monitoring, and prognosis of PCC. This review was conducted by searching PubMed and Scopus databases for keywords and synonyms related to DHTs, Smart Healthcare Systems, and PCC based on the World Health Organization definition. Articles published from 1 January 2020 to 21 May 2024 were screened for eligibility based on predefined inclusion criteria, and the PRISMA framework was used to report the findings from the retained studies. Our search identified 377 studies, but we retained 23 studies that used DHTs, artificial intelligence (AI), and infodemiology to diagnose, estimate prevalence, predict, treat, and monitor PCC. Notably, a few interventions used infodemics to identify the clinical presentations of the disease, while most utilized Electronic Health Records and AI tools to estimate diagnosis and prevalence. However, we found that AI tools were scarcely used for monitoring symptoms, and studies involving SHS were non-existent in low- and middle-income countries (LMICs). These findings show several DHTs used in healthcare, but there is an urgent need for further research in SHS for complex health conditions, particularly in LMICs. Enhancing DHTs and integrating AI and infodemiology provide promising avenues for managing epidemics and related complications, such as PCC.

大流行期间出现了后柯达病症(PCC),促使人们更多使用数字医疗技术(DHT)来管理封锁和医院人满为患的情况。实时跟踪和信息分析对于加强全球研究响应至关重要。本研究旨在绘制现代数字方法在估计 PCC 发病率、预测、诊断、治疗、监测和预后方面的应用图。根据世界卫生组织的定义,本综述通过搜索 PubMed 和 Scopus 数据库中与 DHT、智能医疗系统和 PCC 相关的关键词和同义词进行。根据预定义的纳入标准筛选了 2020 年 1 月 1 日至 2024 年 5 月 21 日期间发表的文章,并采用 PRISMA 框架报告了保留研究的结果。我们的搜索发现了 377 项研究,但保留了 23 项使用 DHT、人工智能 (AI) 和信息病理学来诊断、估计患病率、预测、治疗和监测 PCC 的研究。值得注意的是,少数干预措施使用信息病理学来识别疾病的临床表现,而大多数干预措施则利用电子健康记录和人工智能工具来估计诊断和患病率。然而,我们发现人工智能工具很少被用于监测症状,在中低收入国家(LMICs)也不存在涉及社会和人文科学的研究。这些研究结果表明,有几种 DHT 被用于医疗保健领域,但对于复杂的健康状况,尤其是在低收入和中等收入国家,迫切需要进一步开展社会和人文科学研究。加强 DHTs 并将人工智能与信息病理学相结合,为管理流行病和相关并发症(如 PCC)提供了前景广阔的途径。
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引用次数: 0
Oligoribonucleotide interference-PCR-based methods for the sensitive and accurate detection of KRAS mutations. 基于寡聚核苷酸干扰-PCR 的 KRAS 突变灵敏准确检测方法。
IF 2.5 Q3 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-10-01 eCollection Date: 2024-01-01 DOI: 10.1093/biomethods/bpae071
Hiroaki Fujita, Toshitsugu Fujita, Keinosuke Ishido, Kenichi Hakamada, Hodaka Fujii

Pancreatic cancer is an aggressive malignancy with a poor prognosis. Single-nucleotide mutations in the KRAS gene are detected in the majority of patients with pancreatic ductal adenocarcinoma (PDAC), the most common type of pancreatic cancer. Identifying KRAS mutations by liquid biopsy could be effective for detecting de novo and recurrent PDAC; however, sensitive and accurate detection remains challenging. We examined the utility of oligoribonucleotide interference-PCR (ORNi-PCR) followed by real-time PCR or droplet digital PCR (ddPCR) for detecting KRAS single-nucleotide mutations by liquid biopsy. A model of cell-free DNA was used to demonstrate that the ORNi-PCR-based methods are more sensitive and accurate for detecting KRAS mutant DNA than conventional real-time PCR or ddPCR. ORNi-PCR-based methods could be useful for early detection of de novo and recurrent PDAC by liquid biopsy for cancer diagnosis.

胰腺癌是一种侵袭性恶性肿瘤,预后较差。大多数胰腺导管腺癌(PDAC)患者都能检测到 KRAS 基因的单核苷酸突变,PDAC 是最常见的胰腺癌类型。通过液体活检鉴定 KRAS 基因突变可有效检测新发和复发性 PDAC;然而,灵敏而准确的检测仍具有挑战性。我们研究了先用寡核苷酸干扰PCR(ORNi-PCR)再用实时PCR或液滴数字PCR(ddPCR)通过液体活检检测KRAS单核苷酸突变的实用性。通过无细胞DNA模型证明,基于ORNi-PCR的方法在检测KRAS突变DNA方面比传统的实时PCR或ddPCR更灵敏、更准确。基于 ORNi-PCR 的方法可用于通过液体活检早期检测新发和复发性 PDAC,从而进行癌症诊断。
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引用次数: 0
Optimization and application of digital droplet PCR for the detection of SARS-CoV-2 in saliva specimen using commercially available kit. 使用市售试剂盒优化和应用数字液滴 PCR 检测唾液样本中的 SARS-CoV-2
IF 2.5 Q3 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-09-23 eCollection Date: 2024-01-01 DOI: 10.1093/biomethods/bpae068
Maria M M Kaisar, Helen Kristin, Fajar A Wijaya, Clarissa Rachel, Felicia Anggraini, Soegianto Ali

The coronavirus disease-19 pandemic has resulted in a significant global health crisis, causing hundreds of millions of cases and millions of deaths. Despite being declared endemic, SARS-CoV-2 infection continues to pose a significant risk, particularly for immunocompromised individuals, highlighting the need for a more sensitive and specific detection. Reverse transcription digital droplet polymerase chain reaction (RT-ddPCR) possesses a sensitive and absolute quantification compared to the gold standard. This study is the first to optimize RT-ddPCR for detecting SARS-CoV-2 in saliva specimens using a commercially available RT-qPCR kit. Optimization involved the assessment of the RT-ddPCR reaction mixture, annealing temperature adjustments, and validation using 40 stored saliva specimens. RT-qPCR was used as a reference method in this study. Compatibility assessment revealed that ddPCR Supermix for Probes (no dUTP) was preferable with an optimal annealing temperature of 57.6°C. Although a 25% higher primer/probe concentration provides a higher amplitude in droplet separation of positive control, the number of copy numbers decreased. An inverse correlation between Ct value and copy number concentration was displayed, presenting that the lower the Ct value, the higher the concentration, for the N and E genes with r2 values of 0.98 and 0.85, respectively. However, ORF1ab was poorly correlated (r2 of 0.34). The sensitivity of targeted and E genes was 100% and 93.3%, respectively; as for the specificity, the percentage ranged from 80.8% to 91.3%. This study implicates the applicability of a modified method in the ddPCR platform for similar types of pathogens using saliva specimens.

冠状病毒疾病-19 大流行导致了重大的全球健康危机,造成数亿病例和数百万人死亡。尽管已被宣布为地方性流行病,但 SARS-CoV-2 感染仍构成重大风险,尤其是对免疫力低下的人来说,这突出表明需要一种更灵敏、更特异的检测方法。与金标准相比,反转录数字液滴聚合酶链反应(RT-ddPCR)具有灵敏和绝对定量的特点。本研究首次使用市售 RT-qPCR 试剂盒对 RT-ddPCR 进行优化,以检测唾液样本中的 SARS-CoV-2 病毒。优化工作包括评估 RT-ddPCR 反应混合物、调整退火温度以及使用 40 份储存的唾液标本进行验证。本研究将 RT-qPCR 用作参考方法。兼容性评估显示,ddPCR Supermix for Probes(不含 dUTP)最佳退火温度为 57.6°C。虽然引物/探针浓度提高 25% 可使阳性对照的液滴分离振幅增大,但拷贝数却减少了。对于 N 和 E 基因,Ct 值与拷贝数浓度之间呈反相关,即 Ct 值越低,浓度越高,r2 值分别为 0.98 和 0.85。然而,ORF1ab 的相关性较差(r2 为 0.34)。目标基因和 E 基因的灵敏度分别为 100%和 93.3%;特异性则介于 80.8% 和 91.3% 之间。这项研究表明,ddPCR 平台中的改进方法适用于利用唾液标本检测类似类型的病原体。
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引用次数: 0
An efficient protocol for the extraction of pigment-free active polyphenol oxidase and soluble proteins from plant cells. 从植物细胞中提取不含色素的活性多酚氧化酶和可溶性蛋白质的高效方案。
IF 2.5 Q3 BIOCHEMICAL RESEARCH METHODS Pub Date : 2024-09-19 eCollection Date: 2024-01-01 DOI: 10.1093/biomethods/bpae067
Seyit Yuzuak, De-Yu Xie

The elimination of brownish pigments from plant protein extracts has been a challenge in plant biochemistry studies. Although numerous approaches have been developed to reduce pigments for enzyme assays, none has been able to completely remove pigments from plant protein extracts for biochemical studies. A simple and effective protocol was developed to completely remove pigments from plant protein extracts. Proteins were extracted from red anthocyanin-rich transgenic and greenish wild-type tobacco cells cultured on agar-solidified Murashige and Skoog medium. Protein extracts from these cells were brownish or dark due to the pigments. Four approaches were comparatively tested to show that the diethylaminoethyl (DEAE)-Sephadex anion exchange gel column was effective in completely removing pigments to obtain transparent pigment-free protein extracts. A Millipore Amicon® Ultra 10K cut-off filter unit was used to effectively desalt proteins. Moreover, the removal of pigments significantly improved the measurement accuracy of total soluble proteins. Furthermore, enzymatic assays using catechol as a substrate coupled with high-performance liquid chromatography analysis demonstrated that the pigment-free proteins not only showed polyphenol oxidase (PPO) activity but also enhanced the catalytic activity of PPO. Taken together, this protocol is effective for extracting pigment-free plant proteins for plant biochemistry studies. A simple and effective protocol was successfully developed to not only completely and effectively remove anthocyanin and polyphenolics-derived quinone pigments from plant protein extracts but also to decrease the effects of pigments on the measurement accuracy of total soluble proteins. This robust protocol will enhance plant biochemical studies using pigment-free native proteins, which in turn increase their reliability and sensitivity.

在植物生物化学研究中,如何去除植物蛋白质提取物中的褐色色素一直是一个难题。虽然已经开发出许多方法来减少酶测定中的色素,但还没有一种方法能够完全去除植物蛋白提取物中的色素,用于生化研究。我们开发了一种简单有效的方案来完全去除植物蛋白质提取物中的色素。从琼脂固化的 Murashige 和 Skoog 培养基上培养的富含红色花青素的转基因烟草细胞和偏绿色的野生型烟草细胞中提取蛋白质。从这些细胞中提取的蛋白质因色素而呈褐色或深色。对四种方法进行了比较试验,结果表明二乙胺基乙基(DEAE)-Sephadex 阴离子交换凝胶柱能有效地完全去除色素,从而获得不含色素的透明蛋白质提取物。使用 Millipore Amicon® Ultra 10K 截流过滤装置可有效地对蛋白质进行脱盐。此外,色素的去除大大提高了总可溶性蛋白质的测量精度。此外,以儿茶酚为底物进行的酶测定和高效液相色谱分析表明,不含色素的蛋白质不仅具有多酚氧化酶(PPO)活性,而且还增强了 PPO 的催化活性。综上所述,该方案可有效提取无色素植物蛋白用于植物生物化学研究。研究人员成功开发了一种简单有效的方案,不仅能完全有效地去除植物蛋白质提取物中的花青素和多酚衍生醌类色素,还能降低色素对总可溶性蛋白质测量精度的影响。这种稳健的方案将加强使用不含色素的原生蛋白质进行的植物生化研究,从而提高研究的可靠性和灵敏度。
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Biology Methods and Protocols
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