Journal of virological methods最新文献_第2页

Comparing methods to detect cellular proteins on the surface of HIV-1 virions.

IF 2.2 4区医学

Journal of virological methods Pub Date : 2024-12-06 DOI: 10.1016/j.jviromet.2024.115096

Deepa Chaphekar, Claire Fernandes, Arvin T Persaud, Christina Guzzo

{"title":"Comparing methods to detect cellular proteins on the surface of HIV-1 virions.","authors":"Deepa Chaphekar, Claire Fernandes, Arvin T Persaud, Christina Guzzo","doi":"10.1016/j.jviromet.2024.115096","DOIUrl":"10.1016/j.jviromet.2024.115096","url":null,"abstract":"<p><p>The surface of HIV-1 is embedded with numerous host-derived proteins. Characterizing these proteins can enhance knowledge of virus biology and potentially identify novel therapeutic targets. As many of these proteins are present in low abundance on virion surfaces, their identification can be hindered by inherent variables in the methods employed to detect them, including their varying assay sensitivities, sample processing, quantitative capacity, and experimental reproducibility. Here, we have compared the quantification of virion-incorporated proteins using conventional virus immunocapture assays and western blotting, alongside an emerging technique called flow virometry (FV). Using four different pseudovirus models that each express a human protein of interest (CD14, CD38, CD59 and CD162), we compared four experimental techniques for their ability to reliably quantify the incorporation of those four proteins onto virion surfaces. Our results shed light on the advantages and caveats of each technique for detecting virion-incorporated proteins and highlight the breadth in quantification for each technique under different experimental conditions. Protein detection with (FV) provided distinct advantages as it enabled highly reproducible quantifications, had the lowest sample requirements and reagent costs, and minimal hands-on experimental time. We additionally highlight some important considerations in experimental design when studying virion-incorporated proteins, such as the effect of different antibody clones, assay incubation times, and contributions of extracellular vesicles. Most importantly, our data illustrate the importance of using a combination of orthogonal approaches to detect virus-associated proteins, to enable reliable and reproducible quantification that accounts for individual assay biases.</p>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":" ","pages":"115096"},"PeriodicalIF":2.2,"publicationDate":"2024-12-06","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142794968","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

A novel linear B cell epitope of the porcine circovirus type 3 capsid protein identified by phage display technology.

IF 2.2 4区医学

Journal of virological methods Pub Date : 2024-12-05 DOI: 10.1016/j.jviromet.2024.115080

Shu-Qing Yang, Ke Yang, Xin-Ran Li, Yi Zheng, San-Jie Cao, Qi-Gui Yan, Xiao-Bo Huang, Yi-Ping Wen, Qin Zhao, Sen-Yan Du, Yi-Fei Lang, Shan Zhao, Chun Li, Rui Wu

{"title":"A novel linear B cell epitope of the porcine circovirus type 3 capsid protein identified by phage display technology.","authors":"Shu-Qing Yang, Ke Yang, Xin-Ran Li, Yi Zheng, San-Jie Cao, Qi-Gui Yan, Xiao-Bo Huang, Yi-Ping Wen, Qin Zhao, Sen-Yan Du, Yi-Fei Lang, Shan Zhao, Chun Li, Rui Wu","doi":"10.1016/j.jviromet.2024.115080","DOIUrl":"10.1016/j.jviromet.2024.115080","url":null,"abstract":"<p><p>Porcine circovirus type 3 (PCV3) is endemic in swine worldwide and causes reproductive disorders, dermatitis and nephrotic syndrome, and multi-organ inflammation. PCV3 capsid protein (Cap) can self-assemble into virus-like particles (VLPs), and is an ideal candidate for vaccines and diagnostic reagents.In this study, the recombinant PCV3 Cap protein was successfully expressed in E. coli by deleting the nuclear localization sequence (NLS). The PCV3 VLPs were observed by transmission electron microscopy, and its immunogenicity was evaluated in six-week-old female BALB/c mice. A monoclonal antibody was named mAb 2D6, and demonstrated strong reactivity and specificity to PCV3 Cap. The purified mAb 2D6 was further used for bio-panning to select phage expressing specific epitopes from phage-displayed 7 mer-peptide library. A novel linear B-cell epitope, recognized by mAb 2D6, was identified at the amino acid region 47-53 of Cap. The phage peptide sequences were analyzed using multiple sequence alignment and evaluated by peptide ELISA. These results provide insights for developing diagnostic tools and potential vaccines for PCV3.</p>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":" ","pages":"115080"},"PeriodicalIF":2.2,"publicationDate":"2024-12-05","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142786102","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Purification and characterization of kyasanur forest disease virus EDIII domain of major envelope glycoprotein.

IF 2.2 4区医学

Journal of virological methods Pub Date : 2024-12-03 DOI: 10.1016/j.jviromet.2024.115089

Manjima Das, Vivek Kumar, Rishav Madhukalya, Rohit Gupta, Vidushi Agarwal, Shweta Choudhary, Mandar Bhutkar, Shailly Tomar, Dilip Kumar, Rajesh Kumar

{"title":"Purification and characterization of kyasanur forest disease virus EDIII domain of major envelope glycoprotein.","authors":"Manjima Das, Vivek Kumar, Rishav Madhukalya, Rohit Gupta, Vidushi Agarwal, Shweta Choudhary, Mandar Bhutkar, Shailly Tomar, Dilip Kumar, Rajesh Kumar","doi":"10.1016/j.jviromet.2024.115089","DOIUrl":"10.1016/j.jviromet.2024.115089","url":null,"abstract":"<p><p>Current research efforts are underway to create novel approaches for the efficient diagnosis, monitoring, and mitigation of Kyasanur Forest Disease Virus (KFDV) infections. Flavivirus subunit-based vaccines based on envelope glycoprotein EDIII are now in preclinical and clinical research stages. Efficient purification and isolation methods for surface immunogenic viral antigens, including the recombinant envelope immunoglobulin-like domain III (rEDIII) protein, are crucial for the production and manufacturing of promising vaccine candidates that have been extensively assessed in previous literature. Here, we describe a method for high-yield expression, purification, and refolding of a KFDV rEDIII protein from a bacterial expression system. The KFDV rEDIII protein is extracted from the inclusion bodies in urea denaturing buffer followed by nickel-nitrilotriacetic acid (Ni-NTA) affinity chromatography. The purified, denatured KFDV rEDIII protein was subsequently refolded using a step-wise gradient urea dilution via the dialysis method. The circular dichroism and Fourier transform infrared spectroscopy analysis confirms that the refolded KFDV rEDIII maintains the native secondary conformation majorly containing β-strands. Our study provides valuable insights into the design and expression strategies of rEDIII as a novel subunit vaccine candidate against KFDV.</p>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":" ","pages":"115089"},"PeriodicalIF":2.2,"publicationDate":"2024-12-03","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142786117","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Development of a multiplex droplet digital PCR method for detection and differentiation of mpox virus clades 开发用于检测和区分 mpox 病毒支系的多重液滴数字 PCR 方法。

IF 2.2 4区医学

Journal of virological methods Pub Date : 2024-11-26 DOI: 10.1016/j.jviromet.2024.115078

Xiaoyue Chu , Hailong Chen , Rui Wu , Linghao Zhang , Yong Zhang , Hua Xu , Chaofeng Ma

{"title":"Development of a multiplex droplet digital PCR method for detection and differentiation of mpox virus clades","authors":"Xiaoyue Chu , Hailong Chen , Rui Wu , Linghao Zhang , Yong Zhang , Hua Xu , Chaofeng Ma","doi":"10.1016/j.jviromet.2024.115078","DOIUrl":"10.1016/j.jviromet.2024.115078","url":null,"abstract":"<div><h3>Background</h3><div>The current outbreak of mpox has been declared a public health emergency of international concern by the World Health Organization. However, distinguishing symptoms of mpox virus (MPXV) infection from other orthopoxviruses is atypical, necessitating laboratory confirmatory tests to aid in clinical diagnosis. Therefore, rapid and accurate detection and differentiation of various clades of MPXV are imperative.</div></div><div><h3>Objective</h3><div>A multiplex droplet digital PCR (ddPCR) method was developed to detect and differentiate various clades of MPXV with subsequent evaluation of its sensitivity and accessibility through the analysis of 17 clinical samples.</div></div><div><h3>Methods</h3><div>Primers and probes for multiple ddPCR were designed by comparing multiple complete genomes of orthopoxviruses. Primer and probe concentrations, reaction conditions were tentatively optimized on the Biorad QX200 platform. Seventeen clinical samples of MPXV were detected and verified by Sanger sequencing.</div></div><div><h3>Results</h3><div>The established ddPCR method could detect and differentiate MPXV, and the results were consistent with those of Sanger sequencing.</div></div><div><h3>Conclusion</h3><div>Multiplex ddPCR could be used to detect and distinguish different clades of MPXV rapidly and accurately.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"332 ","pages":"Article 115078"},"PeriodicalIF":2.2,"publicationDate":"2024-11-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142739966","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Development and validation of a VP7-specific EIA for determining the potency and stability of inactivated rotavirus vaccine

IF 2.2 4区医学

Journal of virological methods Pub Date : 2024-11-26 DOI: 10.1016/j.jviromet.2024.115079

Sung-Sil Moon , Houping Wang , Kimberly Brown , Yuhuan Wang , Theresa Bessy , Harry B. Greenberg , Baoming Jiang

{"title":"Development and validation of a VP7-specific EIA for determining the potency and stability of inactivated rotavirus vaccine","authors":"Sung-Sil Moon , Houping Wang , Kimberly Brown , Yuhuan Wang , Theresa Bessy , Harry B. Greenberg , Baoming Jiang","doi":"10.1016/j.jviromet.2024.115079","DOIUrl":"10.1016/j.jviromet.2024.115079","url":null,"abstract":"<div><div>To determine the potency of the inactivated rotavirus vaccine (IRV), we developed an enzyme immunoassay (EIA) using a biotin-conjugated RV VP7-specific monoclonal antibody. RV VP7, a pivotal structural protein in the outer capsid layer, governs RV G genotypes and prompts host immune responses, including neutralizing antibodies. This EIA showed high specificity, good linearity, high precision, and high accuracy, with a low limit of detection (LOD) and a limit of quantitation (LOQ) of 0.037 µg/ml RV antigen. The EIA was evaluated and proved suitable for establishing the long-term stability of IRV drug substance (DS) and aluminum-formulated drug product (DP) when stored at −70±10°C and 5±3 °C, respectively. Our results support the use of this EIA to examine the stability and determine the potency, antigen dose, lot-to-lot consistency, and lot release of IRV products. This RV potency assay may serve as an alternative to <em>in vivo</em> potency tests, making it suitable for quality control tests of cGMP IRV lots in clinical trials.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"332 ","pages":"Article 115079"},"PeriodicalIF":2.2,"publicationDate":"2024-11-26","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142748206","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Little influence of DNA quality on the direct sequencing output of non-human primates’ faecal samples DNA质量对非人灵长类粪便样本直接测序结果的影响很小。

IF 2.2 4区医学

Journal of virological methods Pub Date : 2024-11-22 DOI: 10.1016/j.jviromet.2024.115074

Florence C.H. Lee , Frankie T. Sitam , Lu Ping Tan

{"title":"Little influence of DNA quality on the direct sequencing output of non-human primates’ faecal samples","authors":"Florence C.H. Lee , Frankie T. Sitam , Lu Ping Tan","doi":"10.1016/j.jviromet.2024.115074","DOIUrl":"10.1016/j.jviromet.2024.115074","url":null,"abstract":"<div><div>DNA samples selected for long read sequencing (LRS) are routinely required to be ‘pure’ with high DNA concentration. Hence the usefulness of samples with substandard DNA quality for LRS is unknown. We aim to perform de-novo assembly of Adenovirus sequenced from non-human primate (NHP) faeces using the Oxford Nanopore technologies (ONT), an LRS platform. Guided by initial conventional PCR screening, we performed ONT sequencing on 34 Adenovirus positive DNA samples, without prior selection based on faeces freshness level or DNA quality. Non-parametric correlation analysis showed that ONT sequencing outputs is not significantly associated (<em>p</em> > 0.05) with DNA concentrations, faeces freshness levels and the OD ratios of A260/A280 and A260/A230. This indicated that conventional DNA quality parameters may not be the most critical factors in determining the suitability of samples for ONT sequencing. A total of 61.76 % (21/34) of the positive-by-PCR-screening samples yielded Adenovirus reads while 38.24 % (13/34) did not in the PCR-free ONT workflow, although rarefaction analysis showed that sequencing saturation was achieved by all samples. Among the 21 samples with adenovirus reads, ten resulted in at least one Adenovirus contig by the Flye assembler while nine did not and two samples had only a single Adenovirus read. Identity similarity above 90 % in conventional PCR screening may help in selecting ONT positive samples.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"332 ","pages":"Article 115074"},"PeriodicalIF":2.2,"publicationDate":"2024-11-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142695379","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Developing and validating a multiplex hydrolysis probe-based quantitative PCR assay for the detection of four pathogens in chelonians 开发并验证基于水解探针的多重定量 PCR 分析法，用于检测螯虾中的四种病原体。

IF 2.2 4区医学

Journal of virological methods Pub Date : 2024-11-22 DOI: 10.1016/j.jviromet.2024.115077

Maris J. Daleo , Matthew C. Allender

{"title":"Developing and validating a multiplex hydrolysis probe-based quantitative PCR assay for the detection of four pathogens in chelonians","authors":"Maris J. Daleo , Matthew C. Allender","doi":"10.1016/j.jviromet.2024.115077","DOIUrl":"10.1016/j.jviromet.2024.115077","url":null,"abstract":"<div><div>Many wildlife conservation efforts focus on the effects of one pathogen, but for many conservation efforts to be successful, researchers require an understanding of ecological processes that may include multiple co-occurring pathogens. We developed a multiplex quantitative PCR (qPCR) assay to detect four pathogens in eastern box turtles (<em>Terrapene carolina carolina</em>), including frog virus 3 (FV3), <em>Terrapene</em> herpesvirus 1 (TerHV1), box turtle <em>Mycoplasma</em> sp. (BTMyco), and <em>Terrapene</em> adenovirus (TerAdv). TaqMan™ primer probes were designed using previously published assays with four different fluorophores. Multiplex Cq values plotted against singleplex Cq values demonstrated slopes of 0.967, 1.00, 0.980, and 0.973 for TerHV1, TerAdv, FV3, and BTMyco, respectively, and R<sup>2</sup> values of 0.999 for all four pathogens. The assay was highly consistent with the intra-assay variation of all four pathogen targets, ranging from 0.05–1.826 % across all concentrations, while inter-assay variation ranged from 0.031–4.569 % among all four targets at all concentrations. Clinical samples were tested using previously collected samples from eastern box turtles and red-eared sliders (<em>Trachemys scripta elegans</em>) and performed similarly to singleplex assays. This multiplex assay is an effective, time-efficient diagnostic tool to quickly monitor chelonian pathogens by detecting FV3, TerHV1, BTMyco, and TerAdv within a single reaction. A validated and clinically utilized multiplex assay will be beneficial to characterizing a more complex pathogen profile for future chelonian epidemiological studies to better describe pathogen dynamics and their impacts on individual and population health.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"332 ","pages":"Article 115077"},"PeriodicalIF":2.2,"publicationDate":"2024-11-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142695337","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Development of a novel multiplex digital PCR-based method for the detection of HTLV-1 proviral deletion 开发基于多重数字 PCR 的新型方法，用于检测 HTLV-1 前病毒缺失。

IF 2.2 4区医学

Journal of virological methods Pub Date : 2024-11-21 DOI: 10.1016/j.jviromet.2024.115071

Kou Hiraga , Kenta Tezuka , Koh Nagata , Ki-Ryang Koh , Hitomi Nakamura , Yasuko Sagara , Rieko Sobata , Masahiro Satake , Michikazu Tanio , Hiroo Hasegawa , Masumichi Saito , Kiyonori Miura , Takuo Mizukami , Isao Hamaguchi , Madoka Kuramitsu

{"title":"Development of a novel multiplex digital PCR-based method for the detection of HTLV-1 proviral deletion","authors":"Kou Hiraga , Kenta Tezuka , Koh Nagata , Ki-Ryang Koh , Hitomi Nakamura , Yasuko Sagara , Rieko Sobata , Masahiro Satake , Michikazu Tanio , Hiroo Hasegawa , Masumichi Saito , Kiyonori Miura , Takuo Mizukami , Isao Hamaguchi , Madoka Kuramitsu","doi":"10.1016/j.jviromet.2024.115071","DOIUrl":"10.1016/j.jviromet.2024.115071","url":null,"abstract":"<div><div>The human T-cell leukemia virus type 1 (HTLV-1), a retrovirus, integrates into host DNA and causes adult T-cell leukemia/lymphoma (ATL) in some individuals. Two types of defective proviruses, Type 1 and Type 2, are often observed in ATL cells. Here, we developed a 3-plex digital PCR (dPCR) method to detect HTLV-1 proviral deletions by comparing the ratios of copy numbers quantified using specific primer-probes for the LTR, pol, and pX regions. We analyzed HTLV-1-positive asymptomatic carriers (ACs) and AC samples at high risk for developing ATL due to high proviral load (ATL high-risk (HR) ACs) using dPCR. Deletions were identified in 11.8 % (4/34, all Type 1) of ACs and 33.3 % (7/21, Type 1:1, Type 2:6) of ATL HR ACs. dPCR analysis revealed that in three ATL samples, all exhibited Type 1 defective characteristics, and two showed extremely low ratios in the pol region. Clonality analysis of these two samples revealed high monoclonality, indicating monoclonal expansion of ATL cells with defective proviruses. These findings demonstrate that our method effectively detects defective proviruses in both ACs and ATL, providing a valuable tool for understanding the genomic characteristics of proviruses in these conditions.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"332 ","pages":"Article 115071"},"PeriodicalIF":2.2,"publicationDate":"2024-11-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142693053","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Optimization of pan-lyssavirus LN34 assay for streamlined rabies diagnostics by real-time RT-PCR. 通过实时 RT-PCR 优化用于简化狂犬病诊断的泛赖病毒 LN34 检测方法。

IF 2.2 4区医学

Journal of virological methods Pub Date : 2024-11-21 DOI: 10.1016/j.jviromet.2024.115070

Crystal M Gigante, Vaughn Wicker, Kimberly Wilkins, Melanie Seiders, Hui Zhao, Puja Patel, Lillian Orciari, Rene Edgar Condori, Lisa Dettinger, Pamela Yager, Dongxiang Xia, Yu Li

{"title":"Optimization of pan-lyssavirus LN34 assay for streamlined rabies diagnostics by real-time RT-PCR.","authors":"Crystal M Gigante, Vaughn Wicker, Kimberly Wilkins, Melanie Seiders, Hui Zhao, Puja Patel, Lillian Orciari, Rene Edgar Condori, Lisa Dettinger, Pamela Yager, Dongxiang Xia, Yu Li","doi":"10.1016/j.jviromet.2024.115070","DOIUrl":"10.1016/j.jviromet.2024.115070","url":null,"abstract":"<p><p>Reliable, validated diagnostic tests are critical for rabies control in animals and prevention in people. We present a performance assessment and updates to the LN34 real-time RT-PCR assay for rabies diagnosis in postmortem animal brain samples. In two U.S. laboratories during 2017-2022, routine used of the LN34 assay produced excellent diagnostic sensitivity (99.72-100 %) and specificity (99.99-100 %) compared to the direct fluorescence antibody test (DFA). Almost all (>90 %) DFA indeterminate results caused by non-specific or atypical fluorescence were negative by LN34 testing, representing up to 111 cases where unnecessary post-exposure prophylaxis could be avoided. LN34 assay original primer sequences showed low sensitivity for some rare lyssaviruses. Increased primer concentration combined with new primer formulation showed improved performance for impacted lyssaviruses with no loss in performance across diverse rabies virus variants from clinical samples. The updated LN34 and internal control assays were combined into a single-well LN34 multiplexed (LN34M) format, run at half reagent volumes. The LN34M assay showed similar detection of rabies virus to the singleplexed assay with simplified assay set-up, lower cost, and improved quality controls.</p>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":" ","pages":"115070"},"PeriodicalIF":2.2,"publicationDate":"2024-11-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142695431","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0

Accurate vector copy number determination in gammaretroviral vector producer cell clones using triplex digital droplet PCR 利用三重数字液滴聚合酶链式反应准确测定伽马逆转录病毒载体生产细胞克隆中的载体拷贝数。

IF 2.2 4区医学

Journal of virological methods Pub Date : 2024-11-19 DOI: 10.1016/j.jviromet.2024.115075

Tomomine Iida , Yoshiki Nakamura , Katsuhiko Yamamoto , Eiki Maeda , Yukihiro Ikeda

{"title":"Accurate vector copy number determination in gammaretroviral vector producer cell clones using triplex digital droplet PCR","authors":"Tomomine Iida , Yoshiki Nakamura , Katsuhiko Yamamoto , Eiki Maeda , Yukihiro Ikeda","doi":"10.1016/j.jviromet.2024.115075","DOIUrl":"10.1016/j.jviromet.2024.115075","url":null,"abstract":"<div><div>Gammaretroviral vectors are widely used in cellular and gene therapy products because of the availability of stable vector producer cells. Accurately assessing vector copy number (VCN) is critical for selecting appropriate clones to avoid the risks of homologous recombination and complications in mutation detection. Traditional methods such as quantitative polymerase chain reaction (PCR) and Southern blotting have limitations in accuracy and throughput. This study presents a triplex droplet digital PCR (ddPCR) method for analyzing the VCN in gammaretroviral vector producer cells. We designed a universal primer– probe set targeting the packaging signal sequence common to murine leukemia virus– and murine stem cell virus– based gammaretroviral vectors. Two reference genes were selected after karyotyping the PG13 gammaretroviral vector packaging cell line to identify stable chromosomes. The triplex ddPCR assay was optimized and verified using PG13 cells transduced with constructs of different transgene and vector backbones. The assay showed high concordance with Southern blot. Using multiple reference genes ensures accurate and robust VCN assessment, especially in cell lines with chromosomal instability. This method improves the clone selection process for gammaretroviral vector producer cells, accelerates the development of novel cellular and gene therapy products, and ensures their rapid delivery to patients.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"332 ","pages":"Article 115075"},"PeriodicalIF":2.2,"publicationDate":"2024-11-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142682056","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":4,"RegionCategory":"医学","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

引用次数: 0