Journal of virological methods最新文献_第3页

Fully automated sample-to-answer PCR detection of severe fever with thrombocytopenia syndrome virus in cat serum and whole blood 猫血清和全血中发热伴血小板减少综合征病毒的全自动PCR检测。

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2026-05-01 Epub Date: 2026-01-24 DOI: 10.1016/j.jviromet.2026.115353

Shun Sasaki , Keita Ishijima , Ken Maeda , Marifu Yamagishi-Kondoh

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a tick-borne pathogen that causes fatal hemorrhagic disease in humans and animals. In addition to transmission via tick bites, infection can occur through contact with body fluids from infected individuals or animals. This study evaluated the POCKIT™ Central Nucleic Acid Analyzer (POCKIT Central), a fully automated system integrating nucleic acid extraction and insulated isothermal PCR as a rapid diagnostic tool to aid in preventing the spread of SFTSV infection in veterinary medicine. Analytical sensitivity was assessed using four viral strains; all were detectable, although some exhibited reduced sensitivity. In feline whole blood or serum spiked with SFTSV, the system demonstrated sufficient sensitivity for primary diagnosis of cats showing clinical signs of SFTS. Results of analyses of clinical serum samples using POCKIT Central perfectly matched those obtained using conventional reverse transcription–polymerase chain reaction. The POCKIT Central system can detect SFTSV directly in whole blood or serum within 85 min, without requiring complex manual procedures, thus enabling rapid diagnosis and timely therapeutic intervention in veterinary settings, and it also has the potential for application to other animal species.

发热伴血小板减少综合征病毒（SFTSV）是一种蜱传病原体，可在人和动物中引起致命的出血性疾病。除了通过蜱叮咬传播外，感染还可通过接触受感染个体或动物的体液发生。本研究评估POCKIT™中心核酸分析仪（POCKIT Central），这是一种全自动系统，集成了核酸提取和绝缘等温PCR，作为一种快速诊断工具，有助于预防SFTSV感染在兽药中的传播。使用四种病毒株评估分析敏感性；所有的都是可检测的，尽管有些表现出较低的灵敏度。在添加了SFTSV的猫全血或血清中，该系统对出现SFTS临床症状的猫的初步诊断显示出足够的敏感性。POCKIT Central对临床血清样本的分析结果与常规逆转录聚合酶链反应的结果完全吻合。POCKIT Central系统可以在85分钟内直接检测全血或血清中的SFTSV，无需复杂的人工操作，从而在兽医环境中实现快速诊断和及时的治疗干预，并且它也具有应用于其他动物物种的潜力。

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引用次数: 0

Comparative analysis of beet western yellows virus detection methods: Development and validation of a novel real-time RT-PCR assay also targeting beet leaf yellowing virus 甜菜西部黄病毒检测方法的比较分析：一种新的实时RT-PCR检测方法的建立和验证，也针对甜菜叶黄病毒。

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2026-05-01 Epub Date: 2026-01-22 DOI: 10.1016/j.jviromet.2026.115352

Hellin Pierre , Everaert Ellen , Demonty Elisabeth , Richet Pauline , Muhovski Yordan , De Jonghe Kris , Hautier Louis , Steyer Stéphan

Beet western yellows virus (BWYV – Polerovirus BWYV) is a polerovirus associated with yellowing and stunting symptoms in sugar beet and several other crops. Its close relationship with other beet-infecting poleroviruses, has long complicated accurate diagnosis due to serological cross-reactivity and limited molecular data. To overcome these issues, we developed a novel real-time RT-PCR assay enabling the specific and sensitive detection of BWYV and beet leaf yellowing virus (BLYV – Polerovirus BLYV). The assay was designed from a curated genomic dataset to ensure inclusivity and tested in parallel with validated reference methods, including a semi-generic ELISA and a generic polerovirus RT-PCR. Comprehensive validation following the EPPO PM 7/98(5) standard confirmed the method’s high analytical sensitivity, specificity, repeatability, and reproducibility. The assay proved robust to methodological variations and compatible with multiplex diagnostic workflows. Compared with existing approaches, it offers faster, more reliable detection and eliminates cross-reactivity with non-target poleroviruses. This method provides a valuable tool for accurate BWYV surveillance and supports improved management of beet virus yellows in agricultural and phytosanitary contexts.

甜菜西部黄病毒（BWYV - Polerovirus BWYV）是一种与甜菜和其他几种作物的发黄和发育迟缓症状相关的Polerovirus。它与其他感染甜菜的多极病毒关系密切，由于血清学交叉反应性和有限的分子数据，长期以来使准确诊断复杂化。为了克服这些问题，我们开发了一种新的实时RT-PCR检测BWYV和甜菜叶变黄病毒（BLYV - Polerovirus BLYV）的特异性和敏感性。该检测是根据精心设计的基因组数据集设计的，以确保包容性，并与经过验证的参考方法（包括半通用ELISA和通用多病毒RT-PCR）并行测试。按照EPPO PM 7/98(5)标准进行全面验证，证实该方法具有较高的分析灵敏度、特异性、重复性和再现性。该试验证明了方法变化的稳健性，并与多种诊断工作流程兼容。与现有方法相比，它提供了更快、更可靠的检测，并消除了与非靶标多病毒的交叉反应。该方法为准确监测BWYV提供了有价值的工具，并支持在农业和植物检疫环境下改进甜菜病毒黄的管理。

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引用次数: 0

Development of a strand-specific RT-qPCR assay for detecting and quantifying Borna disease virus RNAs in infected cells 建立一种检测和定量感染细胞中博纳病病毒rna的链特异性RT-qPCR方法。

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2026-05-01 Epub Date: 2026-01-25 DOI: 10.1016/j.jviromet.2026.115356

Takehiro Kanda , Shigeki Hattori , Ryo Komorizono , Keizo Tomonaga

Borna disease virus 1 (BoDV-1) is a nonsegmented negative-strand RNA virus (NSV) that persists in the central nervous system of a broad range of mammalian species. Considering the recent reports linking BoDV-1 to fatal encephalitis in humans, a deeper understanding of its viral life cycle is required. Similar to other NSVs, BoDV-1 produces three types of viral RNAs in infected cells: genome, antigenome, and mRNA. Although a conventional reverse transcription quantitative real-time PCR (RT-qPCR) assay is used to detect viral RNAs, this assay cannot distinguish between RNA species because of primer-independent cDNA synthesis during the reverse transcription (RT) reaction. In this study, we developed a strand-specific RT-qPCR (ssRT-qPCR) assay, in which an RT-primer fused with a non-viral tag sequence at the 5’-end is used in the RT reaction. This assay successfully detected and quantified three types of BoDV-1 RNAs distinctively. Furthermore, using this assay, we measured the intracellular kinetics of the BoDV-1 RNA species, revealing that BoDV-1 may control transcription, but not replication, to establish and maintain persistent infection. Overall, this novel ssRT-qPCR assay is a powerful tool for understanding the life cycle of BoDV-1.

博尔纳病病毒1 （BoDV-1）是一种存在于多种哺乳动物中枢神经系统的非节段负链RNA病毒（NSV）。考虑到最近将BoDV-1与人类致命脑炎联系起来的报告，需要更深入地了解其病毒生命周期。与其他nsv类似，BoDV-1在感染细胞中产生三种类型的病毒rna：基因组、抗基因组和mRNA。尽管传统的逆转录定量实时PCR （RT- qpcr）检测方法用于检测病毒RNA，但由于逆转录（RT）反应过程中不依赖于引物的cDNA合成，因此该检测方法无法区分RNA种类。在这项研究中，我们开发了一种链特异性RT- qpcr （ssRT-qPCR）实验，在RT反应中使用在5'端融合非病毒标记序列的RT引物。该试验成功地检测和定量了三种不同类型的BoDV-1 rna。此外，通过该实验，我们测量了BoDV-1 RNA物种的细胞内动力学，揭示了BoDV-1可能控制转录，但不控制复制，以建立和维持持续感染。总之，这种新颖的ssRT-qPCR检测是了解BoDV-1生命周期的有力工具。

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引用次数: 0

A new real-time RT-PCR for the detection of piscine orthoreoviruses PRV-1 and PRV-3 一种检测鱼类正肠病毒PRV-1和PRV-3的实时RT-PCR方法。

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2026-05-01 Epub Date: 2026-01-31 DOI: 10.1016/j.jviromet.2026.115361

Laurane Pallandre, Marine Baud, Emma Rolland, Alexiane Talbodec, Laurent Bigarré

Piscine orthoreoviruses (PRV) are often associated with pathologies affecting various salmonid species, especially farmed species. Both surveillance and research purposes require diagnostic molecular-based tools able to detect the most frequent genotypes, namely PRV-1 and PRV-3. A new one-step reverse-transcription real-time PCR (RT-qPCR) was set up targeting a conserved region of the L1 segment. Based on the French standard NF U47–600, this PCR method proved to be specific to PRV, excluding other salmonid viruses. The assay can detect 1000 copies of a synthetic transcript of PRV-3, pure or mixed with nucleic acids from healthy fish. This assay produced signals similar to those observed using a previously published generic assay targeting a different viral segment. The new assay is reliable and can be used for the detection of PRV-1 and PRV-3 in rainbow trout and Atlantic salmon.

鱼类正呼肠孤病毒（PRV）通常与影响各种鲑科鱼类，特别是养殖鲑科鱼类的病理有关。监测和研究目的都需要基于分子的诊断工具，能够检测最常见的基因型，即PRV-1和PRV-3。建立了一种新的针对L1片段保守区域的一步反转录实时PCR （RT-qPCR）。根据法国标准NF U47-600，该方法对PRV具有特异性，不包括其他鲑科病毒。该检测方法可以检测1000份PRV-3合成转录本，无论是纯的还是与健康鱼类的核酸混合的。该试验产生的信号类似于使用先前发表的针对不同病毒片段的通用试验所观察到的信号。该方法可靠，可用于虹鳟和大西洋鲑鱼中PRV-1和PRV-3的检测。

引用次数: 0

Reporter assays for HPV16 E1/E1C and E2 mRNA splicing using nanoluciferase and secreted luciferase 利用纳米荧光素酶和分泌荧光素酶检测HPV16 E1/E1C和E2 mRNA剪接的报告基因。

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2026-05-01 Epub Date: 2026-01-29 DOI: 10.1016/j.jviromet.2026.115358

Zixiao Jiang , Yani Zhao , Yunji Zheng , Chengyu Hao , Xiaoxu Cui , Johanna Jönsson , Chengjun Wu , Naoko Kajitani , Stefan Schwartz

We have generated two reporter plasmids that either monitor expression of human papillomavirus type 16 (HPV16) E2 mRNAs using splice site SA2709 or HPV16 E1C mRNAs utilizing splice site SA2582. In plasmid pE2sLuc, the entire E2 open reading frame was replaced by the secreted luciferase (sLuc) reporter gene and in the pE1nLuc plasmid the E1/E1C open reading frame was fused in frame with the nanoluciferase (nLuc) reporter gene. We demonstrate that these reporter plasmids can be used to investigate cis-acting RNA elements and trans-acting factor that regulate splicing to the E2 splice site SA2709 and the alternative E1C- splice site SA2582. Furthermore, we generated a stable C33A cell line carrying HPV16 reporter plasmid pE1nLuc. Using this cell line, we showed that splicing to the HPV16 E2 3’-splice site SA2709 could be perturbed by over expression of SR-proteins or by incubation with a pan-Akt kinase inhibitor. These proteins and substances shifted splicing from E2 splice site SA2709 to the upstream HPV16 3’-splice site SA2582 and enhanced nLuc production, thereby demonstrating the utility of the pE1nLuc cell line as a bioassay for HPV16 E2 mRNA splicing.

我们已经生成了两个报告质粒，它们要么利用剪接位点SA2709监测人乳头瘤病毒16型（HPV16） E2 mrna的表达，要么利用剪接位点SA2582监测HPV16 E1C mrna的表达。在质粒pE2sLuc中，整个E2开放阅读框被分泌的荧光素酶（sLuc）报告基因所取代，在质粒pE1nLuc中，E1/E1C开放阅读框与纳米荧光素酶（nLuc）报告基因融合在一起。我们证明这些报告质粒可以用于研究调节E2剪接位点SA2709和E1C剪接位点SA2582剪接的顺式作用RNA元件和反式作用因子。此外，我们还生成了携带HPV16报告质粒pE1nLuc的稳定C33A细胞系。在该细胞系中，我们发现对HPV16 E2 3'-剪接位点SA2709的剪接可以被sr蛋白的过度表达或泛akt激酶抑制剂的孵育所干扰。这些蛋白和物质将E2剪接位点SA2709转移到上游HPV16 3'剪接位点SA2582，并增强了nLuc的产生，从而证明了pE1nLuc细胞系作为HPV16 E2 mRNA剪接的生物测定方法的实用性。

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引用次数: 0

Genetic influence of IFN-γ gene polymorphisms on hepatitis C progression and recovery IFN-γ基因多态性对丙型肝炎进展和恢复的遗传影响

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2026-05-01 Epub Date: 2026-01-06 DOI: 10.1016/j.jviromet.2026.115337

Khair Rafiq, Sanaullah Khan, Muhammad Bar Khan, Muhammad Yaqoob, Muhammad Adnan

Background

Hepatitis C virus (HCV) infection is a significant health concern that leads to liver complications. The interferon gamma (INF-γ) and interferon gamma receptor-1 (IFN-γR1) genes play role in viral infections. This study aims to analyse the genetic influences of IFN-γ and IFN-γR1 genes polymorphisms on HCV infection outcomes.

Methodology

This cross-sectional study comprised 320 subjects, were classified in to two groups; 160 HCV patients and 160 healthy controls. The HCV patients included 50 subjects with spontaneous viral clearance (SVC), 80 patients with chronic hepatitis C (CHC) and 30 subjects with sustained virological responder (SVR). RNA and DNA were extracted from blood samples. HCV detection and genotyping was performed through PCR. The intron-1 of INF-γ and promoter of IFN-γR1 genes were amplified through PCR and sequenced through Sanger’s method.

Results

The frequency of TT, AT and AA genotypes of IFN-γ at + 874 in HCV positive patients was 62(38.75 %), 64(40 %) and 34(21.5 %); in healthy individuals 73(46.0 %), 61(38.12 %) and 26(16.0 %) (p = 0.153). Genotype AT was significantly high in CHC patients 45(57.0 %) while the TT was considerably high in SVC 27(54.0 %) (p < 0.0001). The frequency of TT, TC, and CC genotypes of IFN-γR1 at −56 was 41(25.64 %), 56(35.0 %), and 63(39.36 %) in HCV patients, while 61(38.13 %), 33(20.63 %) and 66(41.25) in healthy controls (p = 0.0001). The TC genotype was detected high in CHC 34(42.60 %) while the CC was observed more in SVC 24(48.0 %) (P < 0.0001).

Conclusion

IFN-γ AT and IFN-γR1 TC genotypes are associated with Hepatitis C susceptibility, while IFN-γ TT and IFN-γR1 CC may influence recovery.

丙型肝炎病毒（HCV）感染是导致肝脏并发症的重要健康问题。干扰素γ (INF-γ)和干扰素γ受体-1 （IFN-γ r1）基因在病毒感染中起作用。本研究旨在分析IFN-γ和IFN-γ r1基因多态性对HCV感染结局的遗传影响。方法横断面研究共纳入320名受试者，分为两组；160名HCV患者和160名健康对照者。HCV患者包括50例自发性病毒清除（SVC）患者，80例慢性丙型肝炎（CHC）患者和30例持续病毒学应答（SVR）患者。从血样中提取RNA和DNA。采用PCR进行HCV检测和基因分型。通过PCR扩增IFN-γ内含子-1和IFN-γ r1基因启动子，并通过Sanger法测序。结果HCV阳性患者IFN-γ + 874的TT、AT和AA基因型频率分别为62（38.75 %）、64（40 %）和34(21.5 %)；73年健康个体(46.0 %),61(38.12 %)和26(16.0 %)(p = 0.153)。基因型AT在CHC患者45中显著高（57.0% %），而TT在SVC患者27中显著高（54.0% %）（p <； 0.0001）。HCV患者TT、TC和CC基因型分别为41（25.64 %）、56（35.0 %）和63(39.36 %)，健康对照组分别为61（38.13 %）、33（20.63 %）和66(41.25)（p = 0.0001）。TC基因型在CHC 34中较高（42.60 %），而CC基因型在SVC 24中较多（48.0 %）（P <； 0.0001）。结论IFN-γ AT和IFN-γ r1 TC基因型与丙型肝炎易感性相关，而IFN-γ TT和IFN-γ r1 CC可能影响康复。

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引用次数: 0

Effects of porcine epidemic diarrhoea virus vaccination timing and route of administration on colostrum antibody levels and prevention of diarrhoea in piglets on a pig farm with porcine epidemic diarrhoea 猪流行性腹泻病毒疫苗接种时间和给药途径对猪流行性腹泻猪场仔猪初乳抗体水平和预防腹泻的影响

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2026-05-01 Epub Date: 2026-01-31 DOI: 10.1016/j.jviromet.2026.115360

Bo Kyu Kang, Dong Suk Min, Deok Il Kim, Seo Hyun Jung, Bo Kyeong Kim, Jin A. Lee, Do Yeon Kim, Jung Young Park, Jin Sik Oh

Piglets rely on passive immunity acquired from maternal-derived antibodies obtained by ingesting colostrum and milk from immunised sows. This study evaluated the differences in colostrum antibody profiles (IgA, IgG, and neutralising antibodies) according to the timing, frequency, and administration route of the porcine epidemic diarrhoea virus (PEDV) vaccination in pigs with confirmed PEDV infection, and to assess the corresponding clinical protective effects of PEDV vaccination in neonatal piglets. Piglets of sows that received two or more vaccinations, including an oral administration of a live attenuated vaccine 1 week before farrowing (3K1L and 5K3K1L groups), showed disappearance of diarrhoea and growth retardation, and exhibited a significantly higher weaning weight. Sows vaccinated with an oral live vaccine approximately 1 week before farrowing had significantly higher IgA levels than those vaccinated 2 weeks before farrowing. Neutralising antibody titres differed among the groups (p = 0.023): the thrice vaccination group (5K3K1L) showed significantly higher titres than the pre-vaccination, once (1K1L and 2K2L), and twice (3K1L) vaccination groups. IgG levels did not differ substantially between the vaccinated and unvaccinated groups. Overall, this study provides scientific evidence linking clinical protection against PEDV to antibody levels, especially IgA, in sow colostrum.

仔猪通过摄取免疫母猪的初乳和乳汁获得母源抗体，从而获得被动免疫。本研究根据猪流行性腹泻病毒（PEDV）确诊猪的接种时间、频率和给药途径，评估初乳抗体谱（IgA、IgG和中和抗体）的差异，并评估新生儿仔猪接种PEDV疫苗的相应临床保护作用。3K1L组和5K3K1L组母猪接种两次或两次以上疫苗，包括在分娩前1周口服减毒活疫苗，仔猪腹泻消失，生长迟缓，断奶体重显著提高。在分娩前约1周接种口服活疫苗的母猪，其IgA水平明显高于分娩前2周接种疫苗的母猪。各组间的中和抗体效价差异显著（p= 0.023）：三次接种组（5K3K1L）的中和抗体效价显著高于接种前、一次接种组（1K1L和2K2L）和两次接种组（3K1L）。IgG水平在接种疫苗组和未接种疫苗组之间没有显著差异。总的来说，本研究提供了将临床对PEDV的保护与母猪初乳中的抗体水平，特别是IgA水平联系起来的科学证据。

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引用次数: 0

Development and application of a real-time RT-PCR assay for the specific detection of influenza D virus D型流感病毒特异性实时RT-PCR检测方法的建立与应用

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2026-05-01 Epub Date: 2026-01-05 DOI: 10.1016/j.jviromet.2026.115338

Jinfeng Wang , Ruiwen Li , Libing Liu , Jieshi Yu , Ning Li , Xincheng Ji , Yue Yang , Xiaoxia Sun , Wanzhe Yuan , Jianchang Wang

Since the influenza D virus (IDV) was first isolated from a swine exhibiting respiratory disease symptoms in the United States in 2011, it has been detected in various animals across more than 20 countries worldwide, including cattle, pigs, goats, sheep, and camels. IDV has emerged as a significant pathogen associated with bovine respiratory disease complex (BRDC). In this study, specific primers and a TaqMan probe were designed based on the conserved region in the PB1 gene to develop a highly specific and efficient real-time RT-PCR assay (RT-qPCR) for IDV. The RT-qPCR assay demonstrated excellent specificity for IDV, without cross-reactions with other common BRDC-associated pathogens. Using in vitro transcribed PB1 RNA as a template, the assay's limit of detection was established at 100 copies/reaction. Furthermore, the developed RT-qPCR assay was evaluated using 417 cattle nasal swab samples collected from 10 different regions in Hebei Province, China, between 2021 and 2022. The results showed that 33 samples (7.91 %) tested positive for IDV. The IDV HEF gene was amplified and sequenced in these 33 positive samples, yielding 10 HEF gene sequences that all belonged to the D/Yama2019 lineage, with nucleotide homology ranging from 98.7 % to 99.1 %. This study successfully developed an RT-qPCR assay for the specific and sensitive detection of IDV, which performed well on the cattle nasal samples. Additionally, it is the first to demonstrate the presence of IDV in cattle herds in Hebei Province, China.

自2011年在美国首次从出现呼吸道疾病症状的猪身上分离出D型流感病毒（IDV）以来，该病毒已在全球20多个国家的各种动物身上被发现，包括牛、猪、山羊、绵羊和骆驼。IDV已成为与牛呼吸道疾病复合体（BRDC）相关的重要病原体。本研究基于PB1基因的保守区设计了特异性引物和TaqMan探针，建立了IDV高特异性、高效的实时RT-PCR检测方法。RT-qPCR检测显示IDV具有良好的特异性，与其他常见brdc相关病原体无交叉反应。以体外转录的PB1 RNA为模板，测定的检测限为100拷贝/反应。此外，利用2021年至2022年间从中国河北省10个不同地区收集的417份牛鼻拭子样本，对所开发的RT-qPCR方法进行了评估。结果显示，33份样本（7.91%）检测出IDV阳性。在33份阳性样本中扩增并测序IDV HEF基因，得到10个HEF基因序列，均属于D/Yama2019谱系，核苷酸同源性为98.7% ~ 99.1%。本研究成功建立了一种特异、灵敏的检测IDV的RT-qPCR方法，该方法在牛鼻样品中表现良好。此外，这是中国河北省首次证实牛群中存在IDV。

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引用次数: 0

Real time PCR-based evaluation of live attenuated lumpy skin disease virus vaccines for immunogenicity and efficacy 基于实时pcr的疙瘩性皮肤病病毒减毒活疫苗免疫原性和有效性评价

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2026-04-01 Epub Date: 2025-12-16 DOI: 10.1016/j.jviromet.2025.115327

Mohamed Samy Abousenna, Heba A. Khafagy, Amal Abd El Moneim Mohamed, Sara El Sawy Ahmed, Fady Abd El Mohsen Shasha, Nermeen Gouda Shafik

Lumpy skin disease (LSD) is a transboundary viral disease of cattle caused by lumpy skin disease virus (LSDV), resulting in substantial economic losses. Control of the disease relies primarily on live attenuated vaccines, making accurate potency assessment essential to ensure protective efficacy. Quantitative real-time PCR (qPCR) has emerged as a rapid and sensitive method for viral detection and quantification. In this study, ten commercial batches of live attenuated LSDV vaccines were evaluated using qPCR, and the molecular titers were compared with conventional tissue culture titration and in vivo protection in a calf challenge model. Tissue culture titration was performed on MDBK cells, qPCR quantified viral genome copy numbers, and in vivo challenge testing involved 56 calves. Statistical analyses, including Pearson and Spearman correlations and linear regression, were used to assess the relationships between qPCR-derived titers, tissue culture results, and protection outcomes. Eight of the ten vaccine batches met the WOAH-recommended potency range (3–4 log₁₀ TCID₅₀/mL), while two batches were suboptimal. qPCR-derived titers showed a strong correlation with tissue culture titers (Pearson’s r = −0.994; p < 0.0001) r = −0.994; p < 0.0001) and with protection outcomes following virulent challenge (r = 0.777; p = 0.008), with lower molecular titers corresponding to reduced protection. These results demonstrate that qPCR is a rapid, sensitive, and reliable surrogate for conventional potency testing, offering biosafe and time-efficient evaluation. Integrating molecular quantification into routine vaccine assessment can improve quality control, reduce reliance on in vivo challenge, facilitate harmonized vaccine deployment, and ultimately enhance field protection against LSD.

肿块性皮肤病（LSD）是由肿块性皮肤病病毒（LSDV）引起的跨界牛病毒性疾病，造成重大经济损失。该病的控制主要依靠减毒活疫苗，因此准确的效力评估对于确保保护效果至关重要。实时荧光定量PCR （qPCR）是一种快速、灵敏的病毒检测和定量方法。本研究采用qPCR技术对10批LSDV减毒活疫苗进行了评价，并与传统的组织培养滴定法和小牛攻毒模型的体内保护作用进行了分子效价比较。对MDBK细胞进行组织培养滴定，qPCR量化病毒基因组拷贝数，并对56头小牛进行体内攻毒试验。统计分析，包括Pearson和Spearman相关性和线性回归，用于评估qpcr衍生滴度、组织培养结果和保护结果之间的关系。十个疫苗批次中有八个达到了世界卫生组织推荐的效力范围（3-4log₁₀TCID₅₀/mL），而两个批次则不理想。qpcr衍生的滴度与组织培养滴度（Pearson’s r = -0.994; p < 0.0001）和毒力攻击后的保护结果（r = 0.777; p = 0.008）有很强的相关性，较低的分子滴度对应于较低的保护。这些结果表明，qPCR是一种快速、灵敏、可靠的替代传统效价检测的方法，具有生物安全性和时效性。将分子定量纳入常规疫苗评估可以改善质量控制，减少对体内挑战的依赖，促进疫苗的协调部署，并最终增强对LSD的野外保护。

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引用次数: 0

Establishment and application of six-plex fluorescent quantitative PCR assay for the detection of calf diarrhea pathogens 犊牛腹泻病原菌六重荧光定量PCR检测方法的建立及应用。

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2026-04-01 Epub Date: 2025-12-08 DOI: 10.1016/j.jviromet.2025.115323

Zheng Niu , Lichen Nie , Jin Yan , Xinru Xu , Xinfeng Hou , Yong Huang , Qian Du , Dewen Tong

Calf diarrhea is a common clinical disease, causing seriously economic losses to the cattle breeding industry. Bovine coronavirus (BCoV), Bovine rotavirus (BRV), Bovine viral diarrhea virus (BVDV), Escherichia coli (E. coli), Salmonella and Clostridium perfringens (C. perfringens) are common pathogens causing calf diarrhea, Diarrhea in calves caused by these six pathogens is clinically similar and most of them have conditions such as mixed or secondary infections. However, the commonly used method of detecting these six pathogens one by one at present is time-consuming and laborious, which seriously restricts the timely and effective prevention and control of calf diarrhea in production practice. This study established a rapid, efficient, and sensitive multiplex fluorescence quantitative PCR detection method, comprising two independent triplex reactions, for the six pathogens causing calf diarrhea. The results show that the drawn standard curve has a good linear relationship, and R² is greater than 0.990. There is no amplification of other common pathogens in calves. The coefficient of variation (CV) of intra-group repetition and inter-group repetition ranged from 0.17 % to 0.70 %. By testing 932 clinical rectal swab samples of diarrheal calves and comparing them with commercial kits, the results showed that the multiplex fluorescence quantitative PCR detection method established in this study had the characteristics of high specificity and high sensitivity. In conclusion, the detection method established in this study can be used for large-scale detection of clinical samples and is of great applied value for the prevention and treatment of calf diarrhea.

犊牛腹泻是临床上常见的疾病，给养牛业造成了严重的经济损失。牛冠状病毒（BCoV）、牛轮状病毒（BRV）、牛病毒性腹泻病毒（BVDV）、大肠杆菌（E. coli）、沙门氏菌和产气荚膜梭菌（C. perfringens）是引起犊牛腹泻的常见病原体，这六种病原体引起的犊牛腹泻在临床上具有相似性，多有混合性或继发性感染等情况。但目前常用的逐一检测这六种病原菌的方法耗时费力，严重制约了生产实践中对犊牛腹泻的及时有效防控。本研究建立了一种快速、高效、灵敏的犊牛腹泻病原菌多重荧光定量PCR检测方法，该方法由两个独立的三重反应组成。结果表明，绘制的标准曲线具有良好的线性关系，R2大于0.990。犊牛中没有其他常见病原体的扩增。组内重复和组间重复的变异系数（CV）为0.17% ~ 0.70%。通过对932例腹泻犊牛临床直肠拭子样本进行检测，并与市售试剂盒进行对比，结果表明，本研究建立的多重荧光定量PCR检测方法具有高特异性和高灵敏度的特点。综上所述，本研究建立的检测方法可用于临床样品的大规模检测，对犊牛腹泻的防治具有重要的应用价值。

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