Analytical biochemistry最新文献_第8页

Mapping transposon insertion sites within bacterial genomes by direct Sanger sequencing 通过直接Sanger测序在细菌基因组中定位转座子插入位点。

IF 2.5 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-09-10 DOI: 10.1016/j.ab.2025.115971

Scott W. Herke , Linda M. Heffernan , William N. Beavers , Basel H. Abuaita

Microbial biomedical research frequently involves mutagenesis by insertion of transposons into the genome. Currently, transposon insertion locations are typically elucidated by Next Generation Sequencing or by Sanger sequencing of PCR products. Here, transposons were located by direct Sanger sequencing of bacterial genomic DNA from both Salmonella enterica (Gram-negative) and Staphylococcus aureus (Gram-positive) cultures. DNA was prepared by shearing to a modal size of ∼2 kb followed by purification by paramagnetic beads. Sequencing reactions involved relatively minor modifications of standard protocols (e.g., extra sequencing polymerase, 75–100 PCR cycles); completed reactions were cleaned by ethanol-EDTA precipitation. Reads were generated on the ABI 3130xl Genetic Analyzer using 50-cm capillary arrays and a run protocol modified for extra sample injection time. Good quality reads of ∼500–800 nt were routinely generated; BLAST results returned nearly 100% matches to genomes in the NCBI database. As implemented, the optimized protocol (post-DNA extraction) could be performed within an 8-h workday (with sequencing results the following day) for ∼$10 (USD) per sequencing reaction. Although this method was developed to locate transposons inserted into bacterial genomes, it seems likely that it can be extended to generate sequence data from even native single-copy genes from small genomes (e.g., <5 Mb).

微生物生物医学研究经常涉及将转座子插入基因组的诱变。目前，转座子插入位置通常是通过下一代测序或PCR产物的Sanger测序来确定的。在这里，转座子是通过直接桑格测序从肠炎沙门氏菌（革兰氏阴性）和金黄色葡萄球菌（革兰氏阳性）培养的细菌基因组DNA定位。通过剪切至约2kb的模态大小制备DNA，然后用顺磁珠纯化。测序反应涉及对标准方案相对较小的修改（例如，额外的测序聚合酶，75-100个PCR循环）；完成的反应用乙醇- edta沉淀法清洗。在ABI 3130xl遗传分析仪上使用50厘米毛细管阵列和修改的运行方案以增加样品注射时间。常规生成约500-800 nt的高质量读数；BLAST结果与NCBI数据库中的基因组几乎100%匹配。优化后的方案（dna后提取）可以在8小时工作日内完成（第二天有测序结果），每个测序反应约10美元。虽然这种方法是为了定位插入细菌基因组的转座子而开发的，但它似乎可以扩展到从小基因组(例如，

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引用次数: 0

Easy reference-guided assembly of nanopore whole plasmid sequencing datasets 易于参考引导组装纳米孔全质粒测序数据集。

IF 2.5 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-09-05 DOI: 10.1016/j.ab.2025.115970

Vinita Sharma , Naiyu Jiang , Yukihiro Nagashima , Hisashi Koiwa

Whole plasmid sequencing (WPS) using Nanopore long read sequencing has emerged as a cost-effective alternative for dideoxy sequencing methods. De novo sequence assembly for large plasmids, however, are not always successful and may produce large assembly gaps. Here we streamlined a reference-guided alignment of WPS nanopore reads using galaxy platform. The process is straightforward and facilitate introduction of WPS as an alternative for researchers who are more used to dideoxy sequencing platform. We demonstrate that our procedure can assemble nanopore sequence reads with different quality datasets and identify variations from reference sequence.

利用纳米孔长读测序的全质粒测序（WPS）已成为二脱氧测序方法的一种具有成本效益的替代方法。然而，大型质粒的从头序列组装并不总是成功的，并且可能产生较大的组装间隙。本研究利用星系平台简化了WPS纳米孔reads的参考导向比对。该过程简单明了，便于引入WPS作为更习惯于二脱氧测序平台的研究人员的替代方案。我们证明了我们的程序可以用不同质量的数据集组装纳米孔序列，并识别参考序列的差异。

引用次数: 0

PreRBP: Interpretable deep learning for RNA-protein binding site prediction with attention mechanism PreRBP：基于注意机制的rna -蛋白结合位点预测的可解释深度学习。

IF 2.5 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-09-04 DOI: 10.1016/j.ab.2025.115968

Huixian Chen , Yun Zuo , Xiangrong Liu , Xiangxiang Zeng , Zhaohong Deng , Jiasong Wu

In the complex process of gene expression and regulation, RNA-binding proteins occupy a pivotal position for RNA. Accurate prediction of RNA-protein binding sites can help researchers better understand RNA-binding proteins and their related mechanisms. And prediction techniques based on machine learning algorithms are both cost-effective and efficient in identifying these binding sites. However, there are some shortcomings in the currently available machine learning methods, such as the input features of the model only consider RNA sequence features, and most of the datasets suffer from class imbalance. To address these issues, this study first uses the publicly available 27 RNA-protein binding site datasets to construct a benchmark dataset. Then, we use RNAshapes and EDeN to obtain the secondary structure of RNA. Higher-order encoding method is used to extract the key information hidden in the RNA sequences and structures. In order to solve the class imbalance problem existing in the dataset, this study utilizes four undersampling algorithms, namely, random undersampling, NearMiss, ENN, and one-sided selection, to remove redundant samples in the negative samples, and lastly, based on Convolutional Neural Network, Bidirectional Long and Short Term Memory Network, this study constructs model PreRBP to predict RNA-protein binding sites.

The experimental results show that the model used in this study has an average AUC of 0.88, which is higher than other existing RNA-protein binding site prediction methods. Also, for the convenience of prediction, an online predictor is developed in this study. The predictor and experimental codes are available at https://github.com/B12-Comet/RBPPrediction.

在复杂的基因表达和调控过程中，RNA结合蛋白对RNA起着举足轻重的作用。准确预测rna -蛋白结合位点有助于研究人员更好地了解rna -蛋白结合及其相关机制。而基于机器学习算法的预测技术在识别这些结合位点方面既经济又有效。然而，目前可用的机器学习方法存在一些不足，例如模型的输入特征只考虑RNA序列特征，大多数数据集存在类不平衡。为了解决这些问题，本研究首先使用公开可用的27个rna -蛋白结合位点数据集构建基准数据集。然后，我们使用RNAshapes和EDeN来获得RNA的二级结构。采用高阶编码方法提取隐藏在RNA序列和结构中的关键信息。为了解决数据集中存在的类不平衡问题，本研究利用随机欠采样、NearMiss、ENN和片面选择四种欠采样算法去除负样本中的冗余样本，最后基于卷积神经网络、双向长短期记忆网络构建PreRBP模型预测rna -蛋白结合位点。实验结果表明，本研究使用的模型的平均AUC为0.88，高于现有的其他rna -蛋白结合位点预测方法。此外，为了便于预测，本研究还开发了一种在线预测器。预测器和实验代码可在https://github.com/B12-Comet/RBPPrediction上获得。

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引用次数: 0

Exploration of endoplasmic reticulum stress-related gene markers in amyotrophic lateral sclerosis: a comprehensive analysis of bioinformatics and machine learning 肌萎缩侧索硬化症内质网应激相关基因标记的探索：生物信息学和机器学习的综合分析

IF 2.5 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-09-04 DOI: 10.1016/j.ab.2025.115969

Jing Wang , Xinmin Li , Fangjie Yang , Pengxue Guo , Chunlin Ren , Zhengfei Duan , Mengyao Bi , Yuting Kong , Yasu Zhang , Jianwei Lu

This study aimed to investigate potential biomarkers related to Endoplasmic reticulum (ER) stress in Amyotrophic lateral sclerosis (ALS) through a comprehensive bioinformatic approach. The gene expression profiles of ALS patients and healthy controls were downloaded from the Gene Expression Omnibus (GEO) database. ER stress-related genes were collected from the MSigDB databases and document literature. The “limma” R package was employed to detect the differentially expressed ER stress-related genes (DE-ERSGs). Three methods of machine learning were applied to select the hub DE-ERSGs. ROC curves were conducted to evaluate model performance. An external dataset was chosen to evaluate the diagnostic capability of hub genes. The CIBERSORT algorithm was used to evaluate the immune cell infiltration characteristics. Additionally, we constructed a systematic ceRNA regulatory network using Cytoscape software and predicted the possible drug candidates using the Enrichr platform. Molecular docking analysis was used to further validate the binding ability of the candidate drug molecules to the hub genes. Six hub DE-ERSGs (ABCA1, CKAP4, TOR1AIP1, MMP9, EDC4, and ALPP) were identified, and the related models performed well. These hub genes were concentrated in multiple pathways and related to various immune cells. Drugs such as nitroglycerin, diazepam, FENRETINIDE, and edaravone exhibited good binding affinity to the hub genes, indicating that they may be promising drugs for the management of ALS. This study revealed the essential role of ER stress in the pathogenesis of ALS from an integrative perspective, providing guidance for the development of new therapeutic targets and diagnostic strategies.

本研究旨在通过综合生物信息学方法研究肌萎缩侧索硬化症（ALS）内质网（ER）应激相关的潜在生物标志物。从gene expression Omnibus （GEO）数据库下载ALS患者和健康对照者的基因表达谱。从MSigDB数据库和文献文献中收集内质网应激相关基因。“limma”R包检测内质网应激相关基因（de - ergs）的差异表达。采用三种机器学习方法选择轮毂de - ergs。采用ROC曲线评价模型的性能。选择一个外部数据集来评估枢纽基因的诊断能力。采用CIBERSORT算法评价免疫细胞浸润特性。此外，我们使用Cytoscape软件构建了一个系统的ceRNA调控网络，并使用enrichment平台预测了可能的候选药物。通过分子对接分析进一步验证候选药物分子与枢纽基因的结合能力。确定了6个hub de - ergs (ABCA1、CKAP4、TOR1AIP1、MMP9、EDC4和ALPP)，相关模型表现良好。这些枢纽基因集中在多种途径中，与多种免疫细胞有关。硝酸甘油、地西泮、芬拉啶和依达拉奉等药物与中枢基因表现出良好的结合亲和力，表明它们可能是治疗ALS的有希望的药物。本研究从综合角度揭示了内质网应激在ALS发病机制中的重要作用，为开发新的治疗靶点和诊断策略提供指导。

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引用次数: 0

Mismatch-sensitive DNA hybridization controlled by inchworm-type peptide nucleic acid–PEG conjugates 尺蠖型肽核酸- peg偶联物控制错配敏感DNA杂交

IF 2.5 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-09-04 DOI: 10.1016/j.ab.2025.115967

Toshihiko Sakurai , Yusuke Hamashita , Takahiro Shibata

The duplex-forming behavior of an inchworm-type PNA–PEG conjugate (i-PPc), engineered for the selective recognition of point mutations in DNA, was assessed through thermodynamic analysis employing UV melting curves and circular dichroism spectroscopy. The i-PPc demonstrated the ability to form stable duplexes exclusively with fully complementary DNA sequences, while no hybridization with single-base mismatched sequences. This binary on/off hybridization behavior was maintained even under physiologically relevant conditions (37 °C), thereby illustrating the exceptional point mutation discrimination capability of i-PPc. The behavior observed can be ascribed to the distinctive structure of i-PPc, wherein two PNA segments, possessing intrinsically different duplex-forming stabilities—high and low—are covalently linked via a flexible PEG linker. The high-stability PNA segment functions as the primary recognition domain for point mutations, thereby defining the sequence specificity of duplex formation. Conversely, the low-stability segment contributes cooperatively to the overall duplex stabilization only when the high-stability segment successfully hybridizes with the target DNA. This cooperative mechanism underlies the sequence-selective duplex formation of i-PPc, highlighting its potential as a highly specific probe for DNA mutation diagnostics. These findings indicate that i-PPc represents a promising platform for point mutation detection and nucleic acid-based molecular diagnostics grounded in DNA hybridization under physiological conditions.

采用紫外熔化曲线和圆二色光谱热力学分析评估了一种用于选择性识别DNA点突变的尺蠖型PNA-PEG共轭物（i-PPc）的双工形成行为。i-PPc能够与完全互补的DNA序列形成稳定的双链，而与单碱基不匹配的序列不杂交。即使在生理相关条件下（37°C），这种二元开/关杂交行为也能保持，从而说明i-PPc具有特殊的点突变识别能力。观察到的行为可以归因于i-PPc的独特结构，其中两个具有本质上不同的双工形成稳定性的PNA片段-高和低-通过柔性PEG连接共价连接。高稳定性的PNA片段作为点突变的主要识别域，从而定义了双链形成的序列特异性。相反，只有当高稳定性片段成功地与目标DNA杂交时，低稳定性片段才有助于整体双工稳定。这种合作机制是i-PPc序列选择性双工形成的基础，突出了其作为DNA突变诊断的高度特异性探针的潜力。这些发现表明，在生理条件下，i-PPc为点突变检测和基于DNA杂交的核酸分子诊断提供了一个很有前景的平台。

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引用次数: 0

Continuous assay for the dNTP triphosphohydrolase of activated SAMHD1 活化SAMHD1的dNTP三磷酸水解酶连续测定。

IF 2.5 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-09-01 DOI: 10.1016/j.ab.2025.115966

Roozbeh Eskandari, Daniel P. Groom, Ryo Tamura, Vern L. Schramm

Sterile alpha motif and histidine-aspartate domain-containing protein 1 (SAMHD1) is the only member of the triphosphoric monoester hydrolase family in humans (dNTP + H₂O → dN + PPPi). The dNTPase activity of SAMHD1 inhibits DNA synthesis, resulting in cell-cycle arrest and restricting viral replication. The complex allosteric regulation mechanism of SAMHD1 and a reaction that lacks a direct spectroscopic signal make its kinetic analysis and inhibitor discovery challenging. We describe a continuous assay for monitoring SAMHD1 phosphatase activity in its activated physiological state. The assay uses a sequential assembly to generate the active tetrameric form of the enzyme. Two phosphatases convert inorganic triphosphate (PPPi) to inorganic phosphate (Pi). The released Pi reacts with the 7-methyl-6-thioguanosine and purine nucleoside phosphorylase to provide a sensitive continuous spectrophotometric assay. The assay is suitable for 96-microwell plate formats to provide a continuous measurement of SAMHD1 activity. The assay is benchmarked with inhibitors of SAMHD1. With a Z-prime value > 0.90, the assay can be used for high-throughput screening of inhibitors for SAMHD1 and characterizing the allosteric or catalytic activity of the new inhibitors.

无菌α基序和组氨酸-天冬氨酸结构域蛋白1 （SAMHD1）是人类三磷酸单酯水解酶家族（dNTP + H2O→dN + PPPi）中唯一的成员。SAMHD1的dNTPase活性抑制DNA合成，导致细胞周期阻滞，限制病毒复制。SAMHD1复杂的变构调节机制和缺乏直接光谱信号的反应给其动力学分析和抑制剂的发现带来了挑战。我们描述了一个连续监测SAMHD1磷酸酶活性在其激活的生理状态。该分析使用顺序组装来产生活性四聚体形式的酶。两种磷酸酶将无机三磷酸（PPPi）转化为无机磷酸（Pi）。释放的Pi与7-甲基-6-硫鸟嘌呤核苷磷酸化酶反应，提供灵敏的连续分光光度测定。该分析适用于96微孔板格式，以提供SAMHD1活性的连续测量。该试验以SAMHD1抑制剂为基准。该试验的Z-prime值为> 0.90，可用于高通量筛选SAMHD1抑制剂，并表征新抑制剂的变构或催化活性。

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引用次数: 0

Boron nitride quantum dot-based fluorescent sensor for carbamazepine determination in exhaled breath condensate 氮化硼量子点荧光传感器测定呼出液中卡马西平

IF 2.5 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-08-26 DOI: 10.1016/j.ab.2025.115964

Saba Ershadi , Maryam Khoubnasabjafari , Vahid Jouyban-Gharamaleki , Elaheh Rahimpour , Abolghasem Jouyban

Carbamazepine is a widely prescribed antiepileptic drug with a narrow therapeutic index, necessitating precise monitoring to avoid toxicity and ensure therapeutic efficacy. This study presents a fluorescence-based nanosensor using boron nitride quantum dots (BNQDs) for the rapid and sensitive detection of carbamazepine in exhaled breath condensate (EBC). BNQDs were prepared via a simple hydrothermal technique and characterized using transmission electron microscopy, dynamic light scattering, energy-dispersive X-ray, and attenuated total reflectance-Fourier transform infrared techniques. The sensor exhibited a concentration-dependent quenching of BNQD fluorescence upon carbamazepine addition, with a linear response range of 0.2–2.4 μg mL⁻¹ and a low detection limit of 0.05 μg mL⁻¹. Stern–Volmer analysis confirmed a dynamic quenching mechanism. The nanosensor also demonstrated high selectivity against common co-administered drugs and was successfully applied to real EBC samples collected from patients receiving carbamazepine therapy. This BNQD-based sensing platform offered a rapid, cost-effective, and user-friendly approach for the non-invasive therapeutic drug monitoring of carbamazepine.

卡马西平是一种广泛使用的抗癫痫药物，治疗指标较窄，需要精确监测，以避免毒性，确保治疗效果。本研究提出了一种利用氮化硼量子点（BNQDs）的荧光纳米传感器，用于快速灵敏地检测呼出液（EBC）中的卡马西平。采用简单的水热法制备了BNQDs，并利用透射电子显微镜、动态光散射、能量色散x射线和衰减全反射-傅里叶变换红外技术对其进行了表征。该传感器在卡马西平的作用下BNQD荧光猝灭具有浓度依赖性，线性响应范围为0.2 ~ 2.4 μ mL−1，低检出限为0.05 μ mL−1。Stern-Volmer分析证实了动态淬火机制。该纳米传感器还显示出对常见药物的高选择性，并成功应用于卡马西平治疗患者的真实EBC样本。这种基于bnqd的传感平台为卡马西平的无创治疗药物监测提供了一种快速、经济、用户友好的方法。

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引用次数: 0

ITGB3 as a promising non-invasive biomarker for type 2 diabetes and diabetic nephropathy ITGB3有望成为2型糖尿病和糖尿病肾病的无创生物标志物

IF 2.5 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-08-25 DOI: 10.1016/j.ab.2025.115965

Seyed Amirhossein Hosseini , Parisa Ajorlou , Hasti Haddadian , Shahla Sohrabipour

The prevalence of Type 2 diabetes mellitus (T2DM) is increasing worldwide and represents a major risk factor for the development of diabetic nephropathy (DN), a severe microvascular complication. Chronic hyperglycemia activates inflammatory and fibrotic signaling pathways, which contribute to kidney damage. Integrins, as transmembrane adhesion receptors, play pivotal roles in regulating inflammation, immune cell trafficking, and insulin resistance. This research focused on identifying non-invasive biomarkers for T2DM and DN using PBMCs. Differentially expressed genes related to diabetes were identified through the analysis of multiple datasets retrieved from the Gene Expression Omnibus, including GSE95849, GSE9006, GSE25724, and GSE159984. ITGB3 was identified as a common gene across these datasets, and its expression in DN was further examined using the GSE142025 dataset. Real-time PCR analysis of PBMC samples revealed a significant upregulation of ITGB3 expression in individuals with DN and T2DM compared to healthy controls. The TF2DNA and miRNASNPv3 databases identified 10 transcription factors and 10 variants of ITGB3 involved in 60 miRNA interactions. Additionally, the DGIdb database revealed 15 drugs potentially regulating ITGB3 expression. These findings underscore the importance of integrin-related pathways in diabetes and suggest ITGB3 as a promising target for future research and therapeutic development.

2型糖尿病（T2DM）的患病率在全球范围内呈上升趋势，是糖尿病肾病（DN）发展的主要危险因素，是一种严重的微血管并发症。慢性高血糖会激活炎症和纤维化信号通路，从而导致肾脏损伤。整合素作为跨膜粘附受体，在调节炎症、免疫细胞运输和胰岛素抵抗中起关键作用。本研究的重点是利用pbmc识别T2DM和DN的非侵入性生物标志物。通过分析从Gene Expression Omnibus检索到的多个数据集，包括GSE95849、GSE9006、GSE25724和GSE159984，鉴定出与糖尿病相关的差异表达基因。ITGB3被鉴定为这些数据集中的共同基因，并使用GSE142025数据集进一步检测其在DN中的表达。PBMC样本的实时PCR分析显示，与健康对照相比，DN和T2DM患者的ITGB3表达显著上调。TF2DNA和miRNASNPv3数据库鉴定了参与60种miRNA相互作用的10个转录因子和10个ITGB3变体。此外，DGIdb数据库还发现了15种可能调节ITGB3表达的药物。这些发现强调了整合素相关通路在糖尿病中的重要性，并表明ITGB3是未来研究和治疗开发的有希望的靶点。

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引用次数: 0

A novel dual-function sustainable method for H2O2 determination and dye biomineralization utilizing peroxidase-like MnFe2O4@CrFe2O4 nanocomposite 利用过氧化物酶样MnFe2O4@CrFe2O4纳米复合材料的新型双功能可持续的H2O2测定和染料生物矿化方法

IF 2.5 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-08-22 DOI: 10.1016/j.ab.2025.115962

Saeed Reza Hormozi Jangi , Masood Ranjoori , Anahita Barghi , Pardis Ahmadi

In this contribution, a novel dual-function sustainable green nanozyme-mediated method for highly sensitive and selective H₂O₂ quantification and reusable rhodamine B biomineralization utilizing highly active MnFe₂O₄@CrFe₂O₄ nanocomposite with synergistic peroxidase-like activity was designed and developed. This method also introduced a sustainable approach for probing the analyte instead of exploiting prevalent carcinogenic nanozyme-based analytical probes, making it absolutely more sustainable than the conventional methods. The hydrogen peroxide biosensor acquired a linear range of 1–100 μM and a very low detection limit of 0.6 μM, along with an inter-day %RSD of 2.74 % and a highly selective response against coexisting materials. Ultimately, the sensor was employed for H₂O₂ quantification in milk, revealing a recovery of 96.1–102.8 %, %RSD = 1.4–3.6 %. Besides, the effective factors on decolorization yield were optimized, providing a high biomineralization yield of 99.4 % at optimal experimental conditions within a short time of 35.0 min. The breakthrough volume, storage stability, and reusability of the nanozymes were assessed, revealing a breakthrough volume of 5.0–1000 mL, a shelf-life of 20 days, and 70 % yield saving after 10 cycles. The method was applied for dye degradation in real water media, including river water, pool water, and tap water, revealing a high yield of over 95.4–99.5 %, %RSD = 1.8–4.2 %.

本文设计并开发了一种新型的双功能可持续绿色纳米酶介导的方法，利用具有协同过氧化物酶样活性的高活性MnFe2O4@CrFe2O4纳米复合材料，用于高灵敏度和选择性的H2O2定量和可重复使用的罗丹明B生物矿化。该方法还引入了一种可持续的方法来探测分析物，而不是利用流行的致癌纳米酶分析探针，使其绝对比传统方法更具可持续性。过氧化氢生物传感器的线性范围为1 ~ 100 μM，检测限为0.6 μM，日间RSD为2.74%，对共存材料具有高选择性响应。最终将该传感器用于牛奶中H2O2的定量，回收率为96.1 ~ 102.8%,%RSD = 1.4 ~ 3.6%。此外，对影响脱色率的因素进行了优化，在最佳实验条件下，在35.0 min的短时间内，生物矿化率达到99.4%。对纳米酶的突破体积、储存稳定性和可重复使用性进行了评估，结果表明，纳米酶的突破体积为5.0-1000 mL，保质期为20天，10次循环后产量节约70%。将该方法应用于河流、池水、自来水等实际水介质中染料的降解，收率达95.4 ~ 99.5%以上，%RSD = 1.8 ~ 4.2%。

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引用次数: 0

Development of species-specific PCR for authentication of medicinal plant Bupleuri Radix and its common adulterants 药用植物柴胡及其常见掺假物种特异性PCR鉴定方法的建立

IF 2.5 4区生物学 Q2 BIOCHEMICAL RESEARCH METHODS

Analytical biochemistry

Pub Date : 2025-08-14 DOI: 10.1016/j.ab.2025.115963

Hui Tian , Xiuna Zhang , Chi Ma , Yidi Yang , Jing Fang , Fanna Qu , Lihong Yang

Bupleuri Radix is a widely used herbal plant, and its official source recognized by Chinese pharmacopoeia is the dried roots of Bupleurum chinense DC. or Bupleurum scorzonerifolium Willd. Although two species share core therapeutic functions, differences in the types of chemical components lead to different clinical applications. Currently, two species have not been distinguished in pharmacopeial standards. Therefore, we hypothesize prescribing them separately based on the pharmacological characteristics may provide better clinical efficacy. Additionally, Bupleurum marginatum Wall. ex DC. var stenophyllum (Wolff) Shan et Y. Li, and Bupleurum bicaule Helm were often misused as Bupleuri Radix. To address above issues, we developed species-specific PCR targeting ITS region. The observed fragments of 252bp, 183bp, 272bp and 165bp correspond to B. chinense, B. scorzonerifolium, B. marginatum var. stenophyllum, and B. bicaule respectively. The PCR exhibited strong discriminative capability, even when analyzing artificial adulterate samples. Furthermore, analysis of commercial samples validated the accuracy of the species information. In summary, the species-specific PCR enables accurate authentication of Bupleuri Radix and common adulterants in the market. It provides a practical tool for quality control of Bupleuri Radix and supports standardization of herbal medicine production.

柴胡是一种应用广泛的中草药植物，其官方来源为中国药典认可的柴胡干根。或柴胡，野柴胡。虽然两种植物共享核心治疗功能，但化学成分类型的差异导致了不同的临床应用。目前，药典标准中未对两种进行区分。因此，我们假设根据其药理特点分别处方可能会有更好的临床疗效。此外，柴胡壁。交货。var stenophyllum (Wolff) Shan et Y. Li和Bupleurum bicaule Helm经常被误用为柴胡根。为了解决上述问题，我们开发了针对ITS区域的物种特异性PCR。252bp、183bp、272bp和165bp的片段分别对应于B. chinense、B. scorzonerifolium、B. marginatum vars . stenophyllum和B. bicaule。即使在分析人工掺假样品时，PCR也表现出较强的判别能力。此外，对商业样本的分析验证了物种信息的准确性。综上所述，物种特异性PCR能够准确鉴别柴胡和市场上常见的掺假物。为柴胡的质量控制提供了实用的工具，为中草药生产的标准化提供了支持。

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引用次数: 0