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.
Objective
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.
Methods
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.
Results
The established ddPCR method could detect and differentiate MPXV, and the results were consistent with those of Sanger sequencing.
Conclusion
Multiplex ddPCR could be used to detect and distinguish different clades of MPXV rapidly and accurately.
{"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}
Pub Date : 2024-11-26DOI: 10.1016/j.jviromet.2024.115079
Sung-Sil Moon , Houping Wang , Kimberly Brown , Yuhuan Wang , Theresa Bessy , Harry B. Greenberg , Baoming Jiang
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 in vivo potency tests, making it suitable for quality control tests of cGMP IRV lots in clinical trials.
{"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}
Pub Date : 2024-11-22DOI: 10.1016/j.jviromet.2024.115074
Florence C.H. Lee , Frankie T. Sitam , Lu Ping Tan
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 (p > 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.
用于长读数测序（LRS）的 DNA 样本通常要求 "纯净"，DNA 浓度高。因此，DNA质量不达标的样本在长读取测序中是否有用还不得而知。我们的目标是利用牛津纳米孔技术（ONT）这一长测序平台，对从非人灵长类动物（NHP）粪便中测序的腺病毒进行重新组装。在初步常规 PCR 筛选的指导下，我们对 34 份腺病毒阳性 DNA 样品进行了 ONT 测序，事先未根据粪便新鲜度或 DNA 质量进行筛选。非参数相关分析表明，ONT测序结果与DNA浓度、粪便新鲜度水平以及A260/A280和A260/A230的OD比值无明显关联（p > 0.05）。这表明传统的 DNA 质量参数可能不是决定样本是否适合 ONT 测序的最关键因素。在无 PCR ONT 工作流程中，61.76%（21/34）的通过 PCR 筛选的阳性样本产生了腺病毒读数，而 38.24%（13/34）的样本没有产生腺病毒读数，尽管稀有度分析表明所有样本都达到了测序饱和。在有腺病毒读数的 21 个样本中，有 10 个样本通过 Flye 汇编器至少产生了一个腺病毒序列，有 9 个样本没有产生，有 2 个样本只有一个腺病毒读数。在常规 PCR 筛选中，相似度超过 90% 可能有助于筛选出 ONT 阳性样本。
{"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}
Pub Date : 2024-11-22DOI: 10.1016/j.jviromet.2024.115077
Maris J. Daleo , Matthew C. Allender
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 (Terrapene carolina carolina), including frog virus 3 (FV3), Terrapene herpesvirus 1 (TerHV1), box turtle Mycoplasma sp. (BTMyco), and Terrapene 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 R2 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 (Trachemys scripta elegans) 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.
{"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}
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.
人类 T 细胞白血病病毒 1 型（HTLV-1）是一种逆转录病毒，可整合到宿主 DNA 中，并在某些人体内引发成人 T 细胞白血病/淋巴瘤（ATL）。在 ATL 细胞中经常可以观察到两种类型的缺陷前病毒，即 1 型和 2 型。在这里，我们开发了一种三重数字 PCR（dPCR）方法，通过比较使用 LTR、pol 和 pX 区域的特异引物探针量化的拷贝数比率来检测 HTLV-1 前病毒缺失。我们使用 dPCR 分析了 HTLV-1 阳性无症状携带者（AC）和因高病毒载量而有可能发展成 ATL 的高风险 AC 样本（ATL 高风险（HR）AC）。dPCR 分析显示，在三个 ATL 样本中，所有样本都表现出 1 型缺陷特征，其中两个样本在 pol 区域表现出极低的比率。对这两个样本进行的克隆性分析表明，其单克隆性很高，表明具有缺陷前病毒的 ATL 细胞是单克隆扩增的。这些研究结果表明，我们的方法能有效检测出 AC 和 ATL 中的缺陷前病毒，为了解这些情况下前病毒的基因组特征提供了宝贵的工具。
{"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}
Pub Date : 2024-11-21DOI: 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
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.
{"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}
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.
{"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}
Pub Date : 2024-11-19DOI: 10.1016/j.jviromet.2024.115069
Anaïs Scohy , Maria A. Argudín , Florence Kabera , Nathalie Olive , Benoît Kabamba Mukadi
CMV infection remains a well-recognized threat in immunocompromised patients and is a major cause of congenital infection. If plasma and whole blood are routinely used as clinical samples for detection of CMV infection and disease, CMV laboratory testing in non-blood samples is becoming more and more relevant to support the diagnosis, prognosis and management of CMV infection. Accurate CMV viral load assessment in various body fluids is therefore essential. We evaluated the performance of the Alinity m CMV assay for detecting and quantifying CMV in plasma, amniotic fluid, bronchoalveolar lavage, cerebrospinal fluid, saliva and urine. Using a commercially available CMV reference panel and CMV culture supernatant, we assessed the linearity and accuracy of the Alinity m CMV assay across the different sample types. Excellent linear correlations (r values > 0.98) and a good accuracy (bias < ± 0.50 Log10 IU/mL and SD < 0.23) were observed. In conclusion, the Alinity m CMV assay is suitable to detect and quantify CMV DNA in plasma but also in all non-plasma samples tested. Random and continuous access capabilities of the Alinity m enable rapid and efficient laboratory detection and quantification of CMV DNA.
CMV 感染在免疫力低下的患者中仍是一个公认的威胁，也是先天性感染的一个主要原因。如果说血浆和全血是检测 CMV 感染和疾病的常规临床样本，那么非血液样本中的 CMV 实验室检测对于支持 CMV 感染的诊断、预后和管理则变得越来越重要。因此，准确评估各种体液中的 CMV 病毒载量至关重要。我们评估了 Alinity m CMV 检测试剂盒检测和定量血浆、羊水、支气管肺泡灌洗液、脑脊液、唾液和尿液中 CMV 的性能。我们使用市售的 CMV 参考面板和 CMV 培养上清，评估了 Alinity m CMV 检测法在不同样本类型中的线性和准确性。我们观察到了极好的线性相关性（r 值 > 0.98）和良好的准确性（偏差 < ± 0.50 Log10 IU/mL，SD < 0.23）。总之，Alinity m CMV 检测试剂盒不仅适用于检测血浆中的 CMV DNA，也适用于检测所有非血浆样本中的 CMV DNA。Alinity m 的随机和连续访问功能可实现快速、高效的 CMV DNA 实验室检测和定量。
{"title":"Evaluation of the Alinity m CMV assay for detecting and quantifying cytomegalovirus DNA in non-plasma samples","authors":"Anaïs Scohy , Maria A. Argudín , Florence Kabera , Nathalie Olive , Benoît Kabamba Mukadi","doi":"10.1016/j.jviromet.2024.115069","DOIUrl":"10.1016/j.jviromet.2024.115069","url":null,"abstract":"<div><div>CMV infection remains a well-recognized threat in immunocompromised patients and is a major cause of congenital infection. If plasma and whole blood are routinely used as clinical samples for detection of CMV infection and disease, CMV laboratory testing in non-blood samples is becoming more and more relevant to support the diagnosis, prognosis and management of CMV infection. Accurate CMV viral load assessment in various body fluids is therefore essential. We evaluated the performance of the Alinity m CMV assay for detecting and quantifying CMV in plasma, amniotic fluid, bronchoalveolar lavage, cerebrospinal fluid, saliva and urine. Using a commercially available CMV reference panel and CMV culture supernatant, we assessed the linearity and accuracy of the Alinity m CMV assay across the different sample types. Excellent linear correlations (r values > 0.98) and a good accuracy (bias < ± 0.50 Log<sub>10</sub> IU/mL and SD < 0.23) were observed. In conclusion, the Alinity m CMV assay is suitable to detect and quantify CMV DNA in plasma but also in all non-plasma samples tested. Random and continuous access capabilities of the Alinity m enable rapid and efficient laboratory detection and quantification of CMV DNA.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"332 ","pages":"Article 115069"},"PeriodicalIF":2.2,"publicationDate":"2024-11-19","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142687357","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}
Pub Date : 2024-11-17DOI: 10.1016/j.jviromet.2024.115062
Azzania Fibriani, Katerina Naisanu, Nicholas Yamahoki, Denti Rizki Kinanti
Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) is the primary cause of the Coronavirus disease 2019 (COVID-19) pandemic, which affects millions of people worldwide with high levels of infectivity and mortality. However, the antibodies developed for COVID-19 research and diagnostics are still limited. Therefore, in this study, we developed polyclonal immunoglobulin (IgY) antibodies from chicken egg yolk targeting multi-epitope antigen of SARS-CoV-2. After immunizing hens with a SARS-CoV-2 multi-epitope peptide, IgY antibodies were isolated from chicken eggs and further characterized using SDS-PAGE and ELISA. The results showed that the IgY antibodies were successfully isolated from egg yolks. The sandwich ELISA results demonstrated that the isolated IgYs could bind to SARS-CoV-2 antigens, both the multi-epitope peptide and the trimeric Spike. Furthermore, the developed polyclonal antibodies could recognize SARS-CoV-2 in human nasopharyngeal swab samples, even at the lowest concentration (dilution at 1:10000). Thus, it can be concluded that the developed polyclonal IgYs were successfully produced and have the potential to be applied in the development of COVID-19 diagnostics.
{"title":"Development of polyclonal chicken egg yolk immunoglobulin Y (IgY) antibodies targeting SARS-CoV-2 multi-epitope antigen","authors":"Azzania Fibriani, Katerina Naisanu, Nicholas Yamahoki, Denti Rizki Kinanti","doi":"10.1016/j.jviromet.2024.115062","DOIUrl":"10.1016/j.jviromet.2024.115062","url":null,"abstract":"<div><div>Severe acute respiratory syndrome-coronavirus-2 (SARS-CoV-2) is the primary cause of the Coronavirus disease 2019 (COVID-19) pandemic, which affects millions of people worldwide with high levels of infectivity and mortality. However, the antibodies developed for COVID-19 research and diagnostics are still limited. Therefore, in this study, we developed polyclonal immunoglobulin (IgY) antibodies from chicken egg yolk targeting multi-epitope antigen of SARS-CoV-2. After immunizing hens with a SARS-CoV-2 multi-epitope peptide, IgY antibodies were isolated from chicken eggs and further characterized using SDS-PAGE and ELISA. The results showed that the IgY antibodies were successfully isolated from egg yolks. The sandwich ELISA results demonstrated that the isolated IgYs could bind to SARS-CoV-2 antigens, both the multi-epitope peptide and the trimeric Spike. Furthermore, the developed polyclonal antibodies could recognize SARS-CoV-2 in human nasopharyngeal swab samples, even at the lowest concentration (dilution at 1:10000). Thus, it can be concluded that the developed polyclonal IgYs were successfully produced and have the potential to be applied in the development of COVID-19 diagnostics.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"331 ","pages":"Article 115062"},"PeriodicalIF":2.2,"publicationDate":"2024-11-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142647708","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}
Pub Date : 2024-11-16DOI: 10.1016/j.jviromet.2024.115073
Caroline L. Ashley , Malik Bloul , Sibel Alca , Lachlan Smith , Wang Jin , David Khoury , Claudio Counoupas , Miles Davenport , James A. Triccas , Megan Steain
A multiplexed, lentivirus-based pseudovirus neutralisation assay (pVNT) was developed for high-throughput measurement of neutralising antibodies (nAbs) against three distinct SARS-CoV-2 spike variants. Intra-assay variability was minimised by optimising the plate layout and determining an optimal percentage transduction for the pseudovirus inoculum. Comparison of EC50 titres between single and multiplexed pVNT assays showed no significant differences, indicating reliability of the multiplexed assay. Evaluation of convalescent human sera confirmed assay robustness, with consistent EC50 titres for variant pseudoviruses relative to the ancestral strain observed across single and multiplexed assays. This multiplexed pVNT provides a reliable tool for assessing nAb responses against SARS-CoV-2 variants and could be used to accelerate preclinical vaccine assessment in preparation for the next coronavirus pandemic.
{"title":"Optimisation of a multiplexed, high throughput assay to measure neutralising antibodies against SARS-CoV-2 variants","authors":"Caroline L. Ashley , Malik Bloul , Sibel Alca , Lachlan Smith , Wang Jin , David Khoury , Claudio Counoupas , Miles Davenport , James A. Triccas , Megan Steain","doi":"10.1016/j.jviromet.2024.115073","DOIUrl":"10.1016/j.jviromet.2024.115073","url":null,"abstract":"<div><div>A multiplexed, lentivirus-based pseudovirus neutralisation assay (pVNT) was developed for high-throughput measurement of neutralising antibodies (nAbs) against three distinct SARS-CoV-2 spike variants. Intra-assay variability was minimised by optimising the plate layout and determining an optimal percentage transduction for the pseudovirus inoculum. Comparison of EC<sub>50</sub> titres between single and multiplexed pVNT assays showed no significant differences, indicating reliability of the multiplexed assay. Evaluation of convalescent human sera confirmed assay robustness, with consistent EC<sub>50</sub> titres for variant pseudoviruses relative to the ancestral strain observed across single and multiplexed assays. This multiplexed pVNT provides a reliable tool for assessing nAb responses against SARS-CoV-2 variants and could be used to accelerate preclinical vaccine assessment in preparation for the next coronavirus pandemic.</div></div>","PeriodicalId":17663,"journal":{"name":"Journal of virological methods","volume":"332 ","pages":"Article 115073"},"PeriodicalIF":2.2,"publicationDate":"2024-11-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"142668436","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}
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