Journal of virological methods最新文献_第3页

Evaluation of the Alinity m CMV assay for detecting and quantifying cytomegalovirus DNA in non-plasma samples 评估用于检测和量化非血浆样本中巨细胞病毒 DNA 的 Alinity m CMV 检测法。

IF 2.2 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2024-11-19 DOI: 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。

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引用次数: 0

Development of polyclonal chicken egg yolk immunoglobulin Y (IgY) antibodies targeting SARS-CoV-2 multi-epitope antigen 针对 SARS-CoV-2 多表位抗原的多克隆鸡卵黄免疫球蛋白 Y (IgY) 抗体的开发。

IF 2.2 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2024-11-17 DOI: 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.

严重急性呼吸系统综合征-冠状病毒-2（SARS-CoV-2）是冠状病毒病 2019（COVID-19）大流行的主要原因，它影响着全球数百万人，感染率和死亡率都很高。然而，为 COVID-19 研究和诊断开发的抗体仍然有限。因此，在本研究中，我们从鸡卵黄中开发了针对 SARS-CoV-2 多表位抗原的多克隆免疫球蛋白（IgY）抗体。用 SARS-CoV-2 多表位肽免疫母鸡后，我们从鸡卵中分离出了 IgY 抗体，并用 SDS-PAGE 和 ELISA 对其进行了进一步鉴定。结果显示，从蛋黄中成功分离出了 IgY 抗体。夹心酶联免疫吸附试验结果表明，分离出的 IgYs 能与 SARS-CoV-2 抗原结合，包括多表位肽和三聚 Spike。此外，即使在最低浓度（1:10000 稀释度）下，所开发的多克隆抗体也能识别人类鼻咽拭子样本中的 SARS-CoV-2。因此，可以得出结论，研制成功的多克隆 IgYs 有潜力应用于 COVID-19 诊断方法的开发。

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引用次数: 0

Optimisation of a multiplexed, high throughput assay to measure neutralising antibodies against SARS-CoV-2 variants 优化多路复用高通量测定法，以测定针对 SARS-CoV-2 变体的中和抗体。

IF 2.2 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2024-11-16 DOI: 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.

开发了一种基于慢病毒的多重伪病毒中和试验（pVNT），用于高通量测定针对三种不同的 SARS-CoV-2 尖峰变体的中和抗体（nAbs）。通过优化平板布局和确定伪病毒接种体的最佳转导百分比，最大限度地减少了测定内变异性。比较单一和多重 pVNT 检测的 EC50 滴度没有发现明显差异，这表明多重检测是可靠的。对康复期人类血清的评估证实了测定的稳健性，在单一和多重测定中观察到的变异伪病毒相对于祖先毒株的 EC50 滴度是一致的。这种多重 pVNT 为评估针对 SARS-CoV-2 变异株的 nAb 反应提供了可靠的工具，可用于加快临床前疫苗评估，为下一次冠状病毒大流行做准备。

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引用次数: 0

Evaluation and comparison of three high throughput assays (Alinity m CMV, Aptima CMV Quant and cobas CMV) for quantifying CMV DNA in plasma samples 评估和比较用于定量血浆样本中 CMV DNA 的三种高通量检测方法（Alinity m CMV、Aptima CMV Quant 和 cobas CMV）。

IF 2.2 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2024-11-15 DOI: 10.1016/j.jviromet.2024.115068

Jodie D’Costa, Doris Chibo, Katherine Soloczynskyj, Mitchell Batty, Rizmina Sameer, Elaine Lee, Thomas Tran, Dimi Mavroulis, Megan Gooey, Eloise Williams, Kathy Jackson

Background

Cytomegalovirus (CMV) can cause symptomatic CMV syndrome or tissue-invasive CMV disease in immunocompromised individuals, including solid-organ transplant and hematopoietic stem cell transplant recipients. In these populations, monitoring of CMV load is essential, assessing both risk of disease and response to antiviral therapy. High throughput commercial assays are currently available for CMV quantitation, but they are often evaluated independently, with few studies comparing these assays. This study evaluated CMV quantitative assays for use with the Roche cobas 6800, Abbott Alinity m and Hologic Panther platforms using stored patient plasma.

Methods

Analytical evaluation was performed using the 1st WHO international standard for human CMV for Nucleic Acid Amplification Techniques (cobas and Alinity m) or the Hologic CMV QC Calibrator 6 (Aptima). Parallel testing of 136 clinical plasma samples was performed across the three platforms.

Results

Linearity for each assay ranged from 98.6 % to 99.96 % and precision and limit of quantitation were as expected with little variation between platforms. 136 clinical plasma samples were evaluated with similar agreement observed between each assay. The greatest positive agreement was between the Aptima Quant and Alinity m assays (95.6 %, 95 % CI 89–98.6 %) and the lowest between the Aptima Quant and cobas assays (94.1 %, 87.4–97.5 %).

Conclusions

All assays were sensitive and accurate when quantifying CMV, and performance across all 3 assays was comparable for monitoring CMV viral loads in patient plasma.

背景：巨细胞病毒（CMV）可导致免疫功能低下者（包括实体器官移植和造血干细胞移植受者）出现无症状的 CMV 综合征或组织侵袭性 CMV 疾病。在这些人群中，监测 CMV 病毒载量至关重要，可评估疾病风险和对抗病毒治疗的反应。目前已有用于 CMV 定量的高通量商业检测方法，但这些检测方法通常是独立评估的，很少有研究对这些检测方法进行比较。本研究使用储存的患者血浆对罗氏 cobas 6800、雅培 Alinity m 和 Hologic Panther 平台的 CMV 定量检测进行了评估：使用核酸扩增技术（cobas 和 Alinity m）或 Hologic CMV QC Calibrator 6 (Aptima)进行分析评估。三个平台同时检测了 136 份临床血浆样本：结果：每种检测方法的线性范围在 98.6% 到 99.96% 之间，精密度和定量限符合预期，不同平台之间的差异很小。对 136 份临床血浆样本进行了评估，发现每种检测方法之间的一致性相似。Aptima Quant 和 Alinity m 检测法之间的正相关性最高（95.6%，95% CI 89-98.6%），Aptima Quant 和 cobas 检测法之间的正相关性最低（94.1%，87.4-97.5%）：结论：在定量检测 CMV 时，所有检测方法的灵敏度和准确度都很高，所有 3 种检测方法在监测患者血浆中 CMV 病毒载量方面的性能相当。

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引用次数: 0

Quantitative and qualitative analysis of seroconversion after one year of vaccination with inactivated SARS-CoV-2 vaccine (CoronaVac®) in healthcare workers: Cross-sectional analytical study "医护人员接种 SARS-CoV-2 灭活疫苗 (CoronaVac®) 一年后血清转换的定量和定性分析：横断面分析研究"。

IF 2.2 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2024-11-15 DOI: 10.1016/j.jviromet.2024.115067

Júlia Gomes da Silva , Viviana Galimberti Arruk , Glaucia Raquel Luciano da Veiga , Luiz Vinícius de Alcantara Sousa , Beatriz da Costa Aguiar Alves , Fernando Luiz Affonso Fonseca , Inneke Marie van der Heijden Natário

A descriptive study was carried out with health professionals in Sao Paulo city. Were included individuals vaccinated with 2 doses of the inactivated vaccine. Demographic, clinical and vaccination information was obtained from professionals with or without comorbidities. Two serological assays were used to identify the presence and quantity of anti-Spike IgG in serum samples. 433 healthy healthcare professionals were included and 58.9 % completed the 4 clinical stages of serological assessment. Among adults and elderly people, 25.2 % had chronic diseases (hypertension 50.5 %, diabetes 10 % and obesity 6.5 %). Most individuals have 95 % protection in the first 3 months after the second dose, and 67.68 % protection after 6 months. Total antibodies ranged from 3 to 10 on the reactivity index, and the anti-RBD IgG levels were high. CoronaVac has a 94 % seroconversion rate after 2 doses and can prevent serious cases and outbreaks of the disease, if used on a large scale.

我们对圣保罗市的医疗专业人员进行了一项描述性研究。研究对象包括接种过两剂灭活疫苗的人员。研究人员从患有或不患有合并症的专业人员那里获得了人口统计学、临床和疫苗接种信息。采用了两种血清学检测方法来确定血清样本中是否存在抗斯派克 IgG 及其数量。共纳入了 433 名健康的医疗保健专业人员，其中 58.9% 完成了血清学评估的 4 个临床阶段。在成年人和老年人中，25.2%患有慢性疾病（高血压50.5%、糖尿病10%和肥胖6.5%）。大多数人在注射第二针后的头 3 个月内有 95% 的保护率，6 个月后有 67.68% 的保护率。总抗体的反应指数从3到10不等，抗RBD IgG水平较高。CoronaVac两剂后的血清转换率为94%，如果大规模使用，可以预防严重病例和疾病爆发。

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引用次数: 0

Mapping and quantification of potato virus A RNA genomes within viral particles and polysomes in infected plant cells 绘制受感染植物细胞中病毒颗粒和多聚体内马铃薯病毒 A RNA 基因组的图谱并对其进行定量。

IF 2.2 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2024-11-15 DOI: 10.1016/j.jviromet.2024.115066

Pinky Dutta, Kristiina Mäkinen

Potato virus A belongs to the genus Potyvirus, a group of single-stranded positive sense RNA viruses infecting crops worldwide. To initiate infection in a host, its genome takes part in different activities, viz., translation, replication, encapsidation during the infection cycle. Extensive research has been carried out to scrutinize the stages of potyviral infection cycle and decipher the strategies it employs to cause disease. Nonetheless, the amount of viral RNA taking part in translation and virion formation, at a given time point, is missing. In this study, we quantified the percentage of viral RNA that exists as virions and those that associates with host polysome, relative to total viral RNA in infected plant tissue. We employed a revised version of immuno-capture reverse transcription PCR and polysome profiling to address our queries. We tested three different coating antibody concentrations and further optimized the immuno-capture reverse transcription PCR protocol to address its limitation of binding and retaining viral particles. Our results indicate that most of the viral RNA (69 %) exists as encapsidated genomes, while 3 % of total viral RNA associates with host polysomes. These findings are crucial for correct interpretation of quantitative translational studies in which correlation must be made between the number of polysome-associated transcripts and the amount of protein synthesized.

马铃薯病毒 A 属于马铃薯病毒属（Potyvirus），是一组感染全球农作物的单链正感 RNA 病毒。为了启动对宿主的感染，其基因组在感染周期中会参与不同的活动，即翻译、复制和封装。为了仔细研究壶状病毒感染周期的各个阶段并破译其致病策略，已经开展了广泛的研究。然而，在特定时间点参与翻译和病毒形成的病毒 RNA 数量却不详。在这项研究中，我们量化了感染植物组织中以病毒形式存在的病毒 RNA 和与宿主多聚体结合的病毒 RNA 占病毒 RNA 总量的百分比。我们采用免疫捕获反转录 PCR 和多聚体分析的修订版来解决我们的疑问。我们测试了三种不同的包被抗体浓度，并进一步优化了免疫捕获反转录 PCR 方案，以解决其在结合和保留病毒颗粒方面的局限性。我们的结果表明，大部分病毒 RNA（69%）以包被基因组的形式存在，而病毒 RNA 总量的 3% 与宿主多聚体结合。这些发现对于正确解释定量转化研究至关重要，因为在定量转化研究中，必须将多聚体相关转录本的数量与合成的蛋白质数量联系起来。

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引用次数: 0

Wastewater sample storage for physicochemical and microbiological analysis 用于理化和微生物分析的废水样本存储。

IF 2.2 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2024-11-14 DOI: 10.1016/j.jviromet.2024.115063

Gordon Webster , Shrinivas Nivrutti Dighe , William B. Perry , Ewan H. Stenhouse , Davey L. Jones , Peter Kille , Andrew J. Weightman

Wastewater-based epidemiology (WBE) is a crucial tool for health and environmental monitoring, providing real-time data on public health indicators by analysis of sewage samples. Ensuring the integrity of these samples from collection to analysis is paramount. This study investigates the effects of different cold-storage conditions on the integrity of wastewater samples, focusing on both microbiological markers (such as extractable nucleic acids, SARS-CoV-2, and crAssphage) and physicochemical parameters (including ammonium, orthophosphate, pH, conductivity, and turbidity). Composite samples from the raw wastewater influent from five wastewater treatment works in South Wales, UK, were stored at 4°C, -20°C, and -80°C, and subjected to up to six freeze-thaw cycles over one year. The study found significant effects of storage temperature on the preservation of certain WBE markers, with the best yield most frequently seen in samples stored at -80°C. However, the majority of WBE markers showed no significant difference between storage at -80°C or at 4°C, demonstrating that it may not always be necessary to archive wastewater samples at ultra-low temperatures, thus reducing CO₂ emissions and laboratory energy costs. These findings underscore the importance of optimized storage conditions to maintain sample integrity, while ensuring accurate and reliable WBE data for public health and environmental monitoring.

污水流行病学（WBE）是健康和环境监测的重要工具，通过分析污水样本提供有关公共健康指标的实时数据。确保这些样本从采集到分析的完整性至关重要。本研究调查了不同冷藏条件对废水样本完整性的影响，重点关注微生物标记物（如可提取核酸、SARS-CoV-2 和 crAssphage）和理化参数（包括氨、正磷酸盐、pH 值、电导率和浊度）。研究人员将英国南威尔士五个污水处理厂的原废水进水复合样本分别储存在 4°C、-20°C 和 -80°C，并在一年内进行了多达六次冻融循环。研究发现，储存温度对某些水生生物碱标记物的保存有明显影响，在-80°C下储存的样本产量最高。不过，大多数水生生物标记物在-80°C 和 4°C 下的保存效果并无明显差异，这表明并不总是有必要在超低温下保存废水样本，从而减少二氧化碳排放和实验室能源成本。这些发现强调了优化存储条件对保持样本完整性的重要性，同时确保为公共卫生和环境监测提供准确可靠的水生生物数据。

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引用次数: 0

Evaluation of the impact of hollow fiber pore size and membrane material on virus concentration using a handheld hollow fiber method 使用手持式中空纤维法评估中空纤维孔径和膜材料对病毒浓度的影响。

IF 2.2 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2024-11-14 DOI: 10.1016/j.jviromet.2024.115065

Shinsuke Higuchi , Tatsuki Satou , Yuki Uchida

Virus concentration using hollow fibers is widely used for vaccine production to maintain viral infectivity with low shear stress and to allow for easier scale-up of production. However, research laboratories often have limited available viral materials at the early stage of vaccine development, making it difficult to find an optimal hollow-fiber. In addition, few research articles have reported on optimizing hollow fiber pore size and membrane structure for virus concentration. In this study, the previously established handheld hollow fiber virus concentration method was modified to reduce the sample volume required and to enable simple and easy hollow fiber screening. The handheld hollow fiber screening method for virus concentration was confirmed to be effective using Zika as a model virus.

使用中空纤维浓缩病毒被广泛用于疫苗生产，以在低剪切应力下保持病毒的感染性，并使生产规模更容易扩大。然而，在疫苗开发的早期阶段，研究实验室可用的病毒材料往往有限，因此很难找到最佳的中空纤维。此外，关于优化中空纤维孔径和膜结构以提高病毒浓度的研究文章也寥寥无几。本研究对之前建立的手持式中空纤维病毒浓缩方法进行了改进，以减少所需的样品量，并实现简单易行的中空纤维筛选。以寨卡病毒为模型，证实了手持式中空纤维病毒浓缩筛选方法的有效性。

引用次数: 0

Validation of a molecular multiplex assay for the simultaneous detection and differentiation of bluetongue virus and epizootic haemorrhagic disease virus in biological samples 验证同时检测和区分生物样本中蓝舌病病毒和流行性出血病病毒的分子多重分析法。

IF 2.2 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2024-11-13 DOI: 10.1016/j.jviromet.2024.115064

Ottavio Portanti, Eugenia Ciarrocchi, Roberta Irelli, Andrea Palombieri, Romolo Salini, Irene Melegari, Maura Pisciella, Simone Pulsoni, Daria Di Sabatino, Massimo Spedicato, Giovanni Savini, Alessio Lorusso

Bluetongue virus (BTV) and epizootic haemorrhagic disease virus (EHDV) are Culicoides-transmitted viruses, circulating in multiple serotypes, that cause two relevant WOAH-listed diseases of ruminants. Following its first identification in Tunisia in 2021, a novel EHDV strain belonging to serotype 8 has been detected in cattle showing BTV-like symptoms in Italy and Spain in 2022, and soon after in Portugal and France. These are European regions with recurrent circulations of different BTV serotypes. Hence, in this study we describe the validation of a TaqMan RT-qPCR panBTV/panEHDV assay, based on well-established primers and probes sets, able to simultaneously detect and distinguish between BTV and EHDV. The implemented assay, characterized by high sensitivity, specificity and good reproducibility, can be successfully applied for the rapid and affordable diagnosis needed in the current epidemiological situation and represents a powerful tool to be employed in surveillance and control strategies with a significant reduction of costs.

蓝舌病病毒（BTV）和附红细胞体出血病病毒（EHDV）是由蜱传播的病毒，以多种血清型流行，可引起两种被列入世界反刍动物卫生组织（WOAH）的相关疾病。继 2021 年在突尼斯首次发现后，2022 年又在意大利和西班牙安达卢西亚的牛群中检测到属于血清型 8 的新型 EHDV 株，这些牛群表现出类似 BTV 的症状，随后不久又在葡萄牙和法国检测到该病毒。这些欧洲地区反复出现不同血清型的 BTV。因此，在本研究中，我们介绍了基于成熟引物和探针组的 TaqMan RT-qPCR 泛 BTV/ 泛 EHDV 检测方法的验证情况，该方法能够同时检测和区分 BTV 和 EHDV。该检测方法灵敏度高、特异性强、重现性好，可成功应用于当前流行病学形势下所需的快速、经济的诊断，并可作为监测和控制策略的有力工具，显著降低成本。

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引用次数: 0

Design, development, and validation of a strand-specific RT-qPCR assay to differentiate replicating versus nonreplicating Rabies virus 设计、开发和验证用于区分复制与非复制狂犬病病毒的链特异性 RT-qPCR 检测方法。

IF 2.2 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2024-11-08 DOI: 10.1016/j.jviromet.2024.115054

Hong Xiang, Chao Hong, Yuan Tian, Yu Gao, Yina Cun, Hongling Zhao, Guilan Li, Yu Chen, Jian Zhou

The Rabies virus, a single-strand RNA virus with a negative-sense polarity, is responsible for causing encephalitis and is a zoonotic disease. If not promptly treated after infection, it has a close to 100 % fatality rate. Similar to other negative-sense polarity single-strand RNA viruses, the Rabies virus requires the creation of a positive-strand RNA intermediate for replication. One approach to identify this replication activity is to detect the complementary strand of the viral RNA genome in suspected infected cells or tissues. The reported Rabies virus RT-qPCR detection methods are designed to detect total viral load in samples without distinguishing between positive- and negative-strand for RNA viruses. As such, in this study, a sensitive Taqman-based strand-specific RT-qPCR assay has been developed to quantitatively detect both the positive- and negative-strand of the Rabies virus. This method demonstrates good reproducibility across a wide dynamic range and exhibits linearity of 8 logs with a lower limit of detection of 10³ copies/μL for the positive-strand and 9 logs with a lower limit of detection of 10² copies/μL for the negative-strand. Notably, it can accurately detect a specific viral RNA strand even in the presence of high levels of the opposite strand, confirming the method’s specificity. In summary, a reliable strand-specific RT-qPCR assay has been developed and validated to differentiate replicating from non-replicating Rabies virus.

狂犬病病毒是一种具有负义极性的单链 RNA 病毒，可引起脑炎，是一种人畜共患病。如果感染后不及时治疗，死亡率接近 100%。与其他负义极性单链 RNA 病毒类似，狂犬病病毒的复制也需要产生正链 RNA 中间体。确定这种复制活动的一种方法是检测可疑感染细胞或组织中病毒 RNA 基因组的互补链。已报道的狂犬病病毒 RT-qPCR 检测方法旨在检测样本中的总病毒载量，而不区分 RNA 病毒的正链和负链。因此，本研究开发了一种灵敏的基于 Taqman 的特异性链 RT-qPCR 检测方法，可定量检测狂犬病病毒的阳性链和阴性链。该方法在宽动态范围内具有良好的重现性，阳性链的线性度为 8 logs，检测下限为 103 copies/μL；阴性链的线性度为 9 logs，检测下限为 102 copies/μL。值得注意的是，即使存在高水平的反向链，它也能准确检测出特定的病毒 RNA 链，从而证实了该方法的特异性。总之，我们开发并验证了一种可靠的特异性链 RT-qPCR 检测方法，可用于区分复制与非复制狂犬病病毒。

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引用次数: 0