BioTechniques最新文献_第4页

Performance of microarray genotyping and massively parallel sequencing for dense SNP profiling of challenged skeletal remains. 微阵列基因分型和大规模平行测序的性能密集SNP分析挑战骨骼遗骸。

IF 2.6 4区工程技术

BioTechniques Pub Date : 2026-01-01 Epub Date: 2026-10-13 DOI: 10.1080/07366205.2026.2746968

Jeff Duty, Bruce Budowle, Steven A Bates, Kristen Mittelman, David Mittelman

{"title":"Performance of microarray genotyping and massively parallel sequencing for dense SNP profiling of challenged skeletal remains.","authors":"Jeff Duty, Bruce Budowle, Steven A Bates, Kristen Mittelman, David Mittelman","doi":"10.1080/07366205.2026.2746968","DOIUrl":"https://doi.org/10.1080/07366205.2026.2746968","url":null,"abstract":"Dense single nucleotide polymorphism (SNP) profiling is central to forensic genetic genealogy (FGG), but challenged forensic samples frequently contain degraded DNA, low quantities of human DNA, and substantial microbial contamination. Although microarrays perform well with high-quality DNA, their performance with challenged forensic specimens is limited. Microarray performance was evaluated on a dilution series of high-quality control DNA and on DNA extracts from 10 skeletal remains that previously yielded successful SNP profiles with massively parallel sequencing (MPS). The skeletal samples represented a range of degradation index values, human DNA quantities, and proportions of human DNA. The high-quality sample series demonstrated robust microarray performance. However, most challenged skeletal samples generated microarray profiles with markedly reduced or inflated heterozygosity, poor signal-to-noise ratios, and large numbers of discordant genotype calls compared with MPS data despite call rates that would traditionally be considered acceptable. Thus, call rate alone can overestimate microarray profile quality with challenged forensic samples; successful microarray genotyping depends on more than human DNA quantity. Because forensic evidence is often limited and irreplaceable, analytical approaches that maximize the likelihood of successful SNP recovery should be prioritized. These findings support MPS as the preferred platform for dense SNP profiling of challenged forensic specimens.","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":"78 1-12","pages":"359-369"},"PeriodicalIF":2.6,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149179354","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

High-resolution molecular typing of vancomycin-resistant Enterococcus faecium from Romania and Bavaria: combining enhanced DNA microarray and next generation sequencing. 罗马尼亚和巴伐利亚耐万古霉素屎肠球菌的高分辨率分子分型：结合增强DNA微阵列和下一代测序。

IF 2.6 4区工程技术

BioTechniques Pub Date : 2026-01-01 Epub Date: 2026-02-02 DOI: 10.1080/07366205.2026.2621054

Ibukun Elizabeth Osadare, Abdinasir Abdilahi, Elke Müller, Mara Lohde, Celia Diezel, Maximilian Collatz, Sascha Braun, Bärbel Kieninger, Anja Eichner, Wulf Schneider-Brachert, Thomas Wellhöfer, Katrin Frankenfeld, Olivia Dorneanu, Stefan Monecke, Ralf Ehricht

{"title":"High-resolution molecular typing of vancomycin-resistant Enterococcus faecium from Romania and Bavaria: combining enhanced DNA microarray and next generation sequencing.","authors":"Ibukun Elizabeth Osadare, Abdinasir Abdilahi, Elke Müller, Mara Lohde, Celia Diezel, Maximilian Collatz, Sascha Braun, Bärbel Kieninger, Anja Eichner, Wulf Schneider-Brachert, Thomas Wellhöfer, Katrin Frankenfeld, Olivia Dorneanu, Stefan Monecke, Ralf Ehricht","doi":"10.1080/07366205.2026.2621054","DOIUrl":"10.1080/07366205.2026.2621054","url":null,"abstract":"Antimicrobial resistance poses a significant challenge for infection control, requiring the development of accurate and high-throughput diagnostic techniques. We expanded and optimized an existing DNA microarray platform for the molecular characterization of vancomycin-resistant Enterococcus (VRE) by incorporating resistance, virulence, species-specific, and typing markers. The enhanced microarray allows for the simultaneous analysis of up to 96 strains, providing detailed genetic profiles of clinical isolates. VRE strains from Romania and Bavaria, Germany, were analyzed, and the results were compared to those obtained using traditional typing methods, such as multilocus sequence typing (MLST). Next-generation sequencing (NGS) was used in parallel to validate the microarray findings and explore genomic relationships. The microarray revealed considerable genetic diversity and potential epidemiological linkages among isolates. A novel hexadecimal-based nomenclature system was introduced for standardized and scalable strain classification. Comparative analysis demonstrated that the array profiles provided greater discriminatory power and practical resolution than MLST. Receiver operating characteristic (ROC) curve analysis of 187 target genes in 220 isolates gave diagnostic sensitivity and specificity of 100%. This integrated approach offers a cost-effective, rapid, and adaptable global VRE surveillance and infection control tool. It provides a practical alternative to conventional typing systems and facilitates early detection of outbreaks and emerging clones.","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":"78 1-12","pages":"1-15"},"PeriodicalIF":2.6,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"146103718","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Strand-specific quantification of L1 ORF0 and related transcripts by multiplex reverse transcription with tagged primers. 用带标记引物多重反转录对L1 ORF0及相关转录物进行链特异性定量。

IF 2.6 4区工程技术

BioTechniques Pub Date : 2026-01-01 Epub Date: 2026-06-08 DOI: 10.1080/07366205.2026.2678311

Kazunari Mizuno, Yutaka Nakachi, Emi Kiyota, Wataru Ukai, Takao Ishii, Eri Hashimoto, Miki Bundo, Kazuya Iwamoto

{"title":"Strand-specific quantification of L1 ORF0 and related transcripts by multiplex reverse transcription with tagged primers.","authors":"Kazunari Mizuno, Yutaka Nakachi, Emi Kiyota, Wataru Ukai, Takao Ishii, Eri Hashimoto, Miki Bundo, Kazuya Iwamoto","doi":"10.1080/07366205.2026.2678311","DOIUrl":"10.1080/07366205.2026.2678311","url":null,"abstract":"Long interspersed nuclear element-1 (LINE-1 or L1) is the only autonomously active retrotransposon in the human genome and produces both sense and antisense transcripts from its 5' untranslated region (5'UTR) of L1Hs, a human-specific L1 subfamily. Among these, ORF0 is an antisense transcript-derived protein implicated in retrotransposition activity, yet its mRNA expression has been difficult to quantify because strand discrimination is required for accurate detection. Here, we developed a strand-specific quantitative polymerase chain reaction (qPCR) method incorporating multiplex gene-specific tagged primers in the reverse transcription (RT) step, which enables simultaneous quantification of antisense ORF0 and sense 5'UTR and ORF2 transcripts. Validation in 5-aza-2'-deoxycytidine-treated neuroblastoma cells confirmed dose-dependent increases in expression of ORF0 and sense transcripts, demonstrating the strand specificity and functionality of this method. Application to human postmortem prefrontal cortex and cerebellum samples revealed stable expression of ORF0 and sense transcripts. This assay provides a robust and scalable tool for the precise quantification of strand-specific L1 transcription, complementing locus-specific analyses, and offering a platform for future studies on the role of ORF0 and L1 antisense transcription.","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":"78 1-12","pages":"253-262"},"PeriodicalIF":2.6,"publicationDate":"2026-01-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"148203929","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Stabilization of human RNA and DNA in stool samples at room temperature. 室温下粪便样本中人类RNA和DNA的稳定。

IF 2.5 4区工程技术

BioTechniques Pub Date : 2025-11-01 Epub Date: 2025-08-18 DOI: 10.1080/07366205.2025.2546762

Regina Preywisch, Roger Løvlie, Moritz Eidens

{"title":"Stabilization of human RNA and DNA in stool samples at room temperature.","authors":"Regina Preywisch, Roger Løvlie, Moritz Eidens","doi":"10.1080/07366205.2025.2546762","DOIUrl":"10.1080/07366205.2025.2546762","url":null,"abstract":"This study examines the stability of human mRNA and DNA in stool samples for noninvasive gastrointestinal disease detection. While stool samples are valuable for diagnosing conditions like inflammatory disorders and colorectal cancer, mRNA instability poses significant challenges, risking false-negative results. To investigate this, 97 stool samples were treated with a specialized stabilizing solution and stored at room temperature, with analyses conducted on Day 1 and Day 15. The research aimed to improve storage protocols for enhanced reliability in mRNA diagnostics, aiding in personalized medicine and biomarker discovery. Results showed variability in total nucleic acid yields, increasing from Day 1 (mean 112 ng/µL) to Day 15 (mean 165 ng/µL), highlighting the benefits of improved homogenization and bacterial lysis. Human DNA remained stable over the 14-day period. For RNA stability, three mRNA markers were analyzed: Carcinoembryonic Antigen (CEACAM5), Prostaglandin-Endoperoxide Synthase 2 (PTGS2) and cortactin (CTTN). Both CEACAM5 (p=0.064) and PTGS2 (p=0.79) maintained stability, while CTTN showed a statistically significant but only modest reduction in expression (p < 0.0001). Overall, the stabilization buffer proved effectiveness in preserving nucleic acids and provided insights into mRNA marker stability over time.","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":" ","pages":"403-411"},"PeriodicalIF":2.5,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"144871263","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Artificial pre-harvest sprouting chamber with moderate humidity better simulates field sprouting for soft winter wheat. 适宜湿度的人工采前发芽室能较好地模拟软冬小麦田间发芽。

IF 2.5 4区工程技术

BioTechniques Pub Date : 2025-11-01 Epub Date: 2025-12-13 DOI: 10.1080/07366205.2025.2601538

Bryan W Penning

{"title":"Artificial pre-harvest sprouting chamber with moderate humidity better simulates field sprouting for soft winter wheat.","authors":"Bryan W Penning","doi":"10.1080/07366205.2025.2601538","DOIUrl":"10.1080/07366205.2025.2601538","url":null,"abstract":"Pre-harvest sprouting, germination of the seed on the spike, causes reduced grain quality and marketability in US wheat. Methods devised to induce and study pre-harvest sprouting vary significantly from one another in procedure, induction time, and/or measurement, and often fail to accurately reflect natural sprouting, which varies over years and locations. An artificial sprouting chamber and protocol with a shorter exposure time and relative humidity more relevant to field conditions was significantly correlated (p < 0.01) with natural pre-harvest sprouting over three years for ten wheat varieties measured by alpha amylase activity. The alpha amylase activities of ten wheat varieties tested under the developed artificial sprouting chamber and protocol were significantly correlated (p = 0.001) with those subjected to overhead irrigated field sprouting tests over three years. Four pairs of the ten varieties were genetically related but showed significantly different sprouting and alpha amylase activity. These varietal pairs may prove useful in studying the genetics of pre-harvest sprouting. While the study was performed in wheat, other susceptible crops such as rice, barley, rye, and sorghum could be tested using this method.","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":" ","pages":"367-375"},"PeriodicalIF":2.5,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145740859","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Optimization of proximity labeling in endothelial cells: overcoming endogenous biotin interference and cost barriers. 内皮细胞邻近标记的优化：克服内源性生物素干扰和成本障碍。

IF 2.6 4区工程技术

BioTechniques Pub Date : 2025-11-01 Epub Date: 2026-01-29 DOI: 10.1080/07366205.2026.2619158

Ying Jiang, Kuizhi Qu, Mengjun Dai, Yan-Ning Rui, Zhen Xu

{"title":"Optimization of proximity labeling in endothelial cells: overcoming endogenous biotin interference and cost barriers.","authors":"Ying Jiang, Kuizhi Qu, Mengjun Dai, Yan-Ning Rui, Zhen Xu","doi":"10.1080/07366205.2026.2619158","DOIUrl":"10.1080/07366205.2026.2619158","url":null,"abstract":"Proximity labeling has become a powerful technique for mapping protein-protein interactions under physiologically relevant conditions, with TurboID offering high enzymatic activity and rapid labeling. However, its application in endothelial systems has been limited, partly due to the presence of endogenous biotin in specialized media, which reduces labeling specificity. Here, we optimized TurboID-mediated proximity labeling in brain microvascular endothelial cells by addressing two key challenges: endogenous biotin interference and cost-effective depletion. We discovered that endothelial cell medium contains substantial biotin levels, which saturate TurboID labeling and obscure the effects of exogenous biotin. Using High Capacity NeutrAvidin™ agarose, we developed a simple and economical method to deplete endogenous biotin, reducing background biotinylation dramatically. We then defined the optimal condition for efficient labeling with minimal toxicity. Using TKS4, a scaffold protein critical for podosome formation, we validated this workflow in brain microvascular endothelial cells and confirmed the efficiency of streptavidin-based enrichment of biotinylated proteins. This study provides a validated and accessible TurboID workflow for endothelial cells, enabling more precise and cost-effective discovery of dynamic protein interaction networks relevant to vascular integrity and disease.","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":"77 11-12","pages":"1-12"},"PeriodicalIF":2.6,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147472587","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Real-time quantification of soluble tartrate-resistant acid phosphatase as a measure of osteoclastogenesis. 可溶性抗酒石酸酸性磷酸酶的实时定量作为破骨细胞发生的测量。

IF 2.6 4区工程技术

BioTechniques Pub Date : 2025-11-01 Epub Date: 2025-12-23 DOI: 10.1080/07366205.2025.2605263

Justin A Roosma, Hammed Gafar, Maxwell T Boruff, Colton R Quinn, Jason W Ashley

引用次数: 0

Benchmarking antibody discovery fidelity and reproducibility with an assay-locked residue fidelity index. 用测定锁定的残留保真度指数对抗体发现保真度和再现性进行基准测试。

IF 2.6 4区工程技术

BioTechniques Pub Date : 2025-11-01 Epub Date: 2026-01-29 DOI: 10.1080/07366205.2026.2619165

Michael P Weiner

{"title":"Benchmarking antibody discovery fidelity and reproducibility with an assay-locked residue fidelity index.","authors":"Michael P Weiner","doi":"10.1080/07366205.2026.2619165","DOIUrl":"10.1080/07366205.2026.2619165","url":null,"abstract":"Reproducibility in antibody discovery is undermined by dropout, paratope degeneracy, and immunogen imprinting. Although guidelines exist, the field lacks a shared, assay-locked metric to benchmark these liabilities across discovery pipelines. We propose the Residue Fidelity Index (RFI), a discovery-stage, within-assay comparative framework that consolidates fidelity liabilities into a single score. Assay-locked means RFI is comparable only within a fully specified assay configuration, uses fixed normalization and weights, and requires co-reporting of component metrics to expose drivers of the composite. RFI is not intended as a universal or optimal standard, but as one implementable example meeting basic benchmarking requirements: defined components, fixed assay context, pre-specified weights, and primary-data disclosure. RFI is reported alongside its components (D, G, I) to visualize reproducibility in parallel with affinity, humanization, and yield. To demonstrate feasibility, we applied RFI in a simulated Epivolve testbed derived from multiple studies, embedding MILKSHAKE (context retention) and Sundae (residue discrimination) validation modules into discovery. Across three campaigns (n = 25 clones), antibodies meeting provisional thresholds (dropout ≤10%, degeneracy ≤0.10, no imprinting) yielded RFI values from 0.610 to 0.982 (mean ± SD 0.922 ± 0.103; I = 1 for clones C04 and C20). These results show how fidelity can be consolidated to prioritize candidates before application-specific cellular validation. Despite limited scope and proprietary immunogens, this work provides a proof of concept for residue-level benchmarking, with per-clone primary values and RFI reported in Supplementary Table S1 for auditability.","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":"77 11-12","pages":"1-9"},"PeriodicalIF":2.6,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC12994182/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"147472559","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Correction. 修正。

IF 2.5 4区工程技术

BioTechniques Pub Date : 2025-11-01 Epub Date: 2025-09-18 DOI: 10.1080/07366205.2025.2563421

引用次数: 0

Comparison of purification protocols for effective large-primer removal between rounds of PCR amplification. 两轮PCR扩增有效去除大引物的纯化方案比较。

IF 2.5 4区工程技术

BioTechniques Pub Date : 2025-11-01 Epub Date: 2025-12-13 DOI: 10.1080/07366205.2025.2598186

Eleni Polatoglou, Abel Bronkhorst, Victor Costina, Catharina Gerhards, Maren Hedtke, Stefan Holdenrieder, Verena Haselmann

{"title":"Comparison of purification protocols for effective large-primer removal between rounds of PCR amplification.","authors":"Eleni Polatoglou, Abel Bronkhorst, Victor Costina, Catharina Gerhards, Maren Hedtke, Stefan Holdenrieder, Verena Haselmann","doi":"10.1080/07366205.2025.2598186","DOIUrl":"10.1080/07366205.2025.2598186","url":null,"abstract":"Effective polymerase chain reaction (PCR) product purification is essential for downstream applications like next-generation sequencing (NGS). While standard protocols remove short primers (20-30 nucleotides [nt]), NGS workflows require efficient removal of larger primers (40-50 nt) to prevent amplification artifacts. This study compared commercial kits (magnetic beads, silica columns, enzymatic degradation) with traditional isopropanol/ethanol precipitation and simple dilution for their ability to remove large primers from a 161-bp KRAS PCR product. Efficacy was assessed by a secondary PCR designed to amplify remaining primers. Optimal yield was achieved with isopropanol precipitation with NH4Ac (2.5-3.0 M) and overnight incubation at 4 to -20 °C. Interestingly, a simple 1:200 dilution showed comparable results. Among commercial kits, magnetic beads demonstrated superior primer removal, as evidenced by a substantially lower concentration of the secondary PCR product (0.3 ng/µL ± 0.23) compared to untreated samples (22.13 ng/µL ± 1.7). For NGS workflows, magnetic beads are the most effective method for removing large primers, while isopropanol precipitation and dilution offer viable, low-cost options, particularly for workflows involving multiple PCR steps.","PeriodicalId":8945,"journal":{"name":"BioTechniques","volume":" ","pages":"355-365"},"PeriodicalIF":2.5,"publicationDate":"2025-11-01","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"145740155","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"工程技术","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0