Journal of virological methods最新文献

Animal models illuminate dengue immunopathogenesis to guide vaccine and therapeutic developments 动物模型阐明登革热的免疫发病机制，以指导疫苗和治疗的发展

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2026-01-16 DOI: 10.1016/j.jviromet.2026.115350

Muhammad Nadir Shabbir , Thuy Linh Duong

Dengue virus (DENV) infection affects over 390 million people annually, and its severe forms, including dengue hemorrhagic fever (DHF) and dengue shock syndrome (DSS), occur as a result of antibody-dependent enhancement (ADE), T-cell dysregulation, and a cytokine storm. This review integrates the results of human immunopathology with those reported by animal models, consolidating the findings in a systematic manner to validate these mechanisms. Research on AG129 interferon receptor-deficient mice consistently demonstrates ADE-mediated viremia and vascular leakage. However, humanized NSG-BLT mice recapitulate the classic antigenic sin of human T cells after heterotypic challenge with DENV. Cynomolgus macaque models are physiologically relevant for testing tetravalent vaccines under near-human circulatory conditions, and diet-induced obese mice are used to phenotype the effects of metabolic comorbidities on IL-1β-driven immunopathology. Data using standardized methods, including Evans blue extravasation assays, multiplex cytokine panels, and endpoints compliant with the ARRIVE 2.0 guidelines for animal research reporting, were associated with human clinical results. ARRIVE 2.0 is a set of guidelines to improve the transparency and reproducibility of animal research. It is worth noting that early experimental studies on the effectiveness and safety of Dengvaxia reported breakthrough viremia in macaque models. The protection provided by the monoclonal antibody C10 was shown to mitigate antibody-dependent enhancement (ADE) in murine models, though it is not associated with the Dengvaxia vaccine. The further development of these animal models will be necessary to expedite the safe development of next-generation dengue vaccines (TAK-003, TV003/TV005), antivirals (favipiravir, sofosbuvir), and immune modulators (IL-6 and TNF-α blockers) for use in endemic areas.

登革热病毒（DENV）感染每年影响超过3.9亿人，其严重形式，包括登革出血热（DHF）和登革休克综合征（DSS），是抗体依赖性增强（ADE）、t细胞失调和细胞因子风暴的结果。本综述将人类免疫病理学结果与动物模型报告的结果相结合，以系统的方式巩固这些发现，以验证这些机制。AG129干扰素受体缺陷小鼠的研究一致表明，ade介导的病毒血症和血管渗漏。然而，人源化NSG-BLT小鼠在DENV异型攻击后再现了人类T细胞的典型抗原性。食蟹猴模型在生理学上与在接近人类循环条件下测试四价疫苗相关，并且使用饮食诱导的肥胖小鼠来表型代谢合并症对il -1β驱动的免疫病理的影响。使用标准化方法的数据，包括Evans蓝色外渗试验、多重细胞因子面板和符合动物研究报告ARRIVE 2.0指南的终点，与人类临床结果相关。ARRIVE 2.0是一套指导方针，旨在提高动物研究的透明度和可重复性。值得注意的是，早期关于登卡夏有效性和安全性的实验研究报道了在猕猴模型中突破病毒血症。在小鼠模型中，单克隆抗体C10提供的保护被证明可以减轻抗体依赖性增强（ADE），尽管它与登卡夏疫苗没有关联。有必要进一步开发这些动物模型，以加快安全开发用于流行地区的下一代登革热疫苗（TAK-003、TV003/TV005）、抗病毒药物（favipiravir、sofosbuvir）和免疫调节剂（IL-6和TNF-α阻断剂）。

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引用次数: 0

Prevalence and ribavirin-mediated elimination of Morchella importuna endornavirus 1 (MiEV1) in cultivated Morchella species in China 利巴韦林介导的羊肚菌内病毒1 （MiEV1）在中国养殖羊肚菌中的流行及消除

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2026-01-12 DOI: 10.1016/j.jviromet.2026.115349

Zhifeng Zhang , Yingjun Zhang , Zengqiang Shang , Xiaoli Wang , Yankui Li , Zhaoyu Wang , Longbo Zhao , Ziyan Peng

Fungal viruses compromise the cultivation of Morchella. We surveyed five mycoviruses (Morchella importuna endornavirus 1 [MiEV1], Morchella importuna endornavirus 2 [MiEV2], Morchella importuna endornavirus 3 [MiEV3], Morchella esculenta fusarivirus 1 [MeFV1], and Morchella esculenta fusarivirus 2 [MeFV2]) in 24 cultivated strains (8 M. importuna, 5 M. septimelata, and 11 M. sextelata) collected across 11 provinces in China and evaluated candidate antiviral agents. Only MiEV1 was detected, with a high incidence of 79.2 % (19/24) across all three Morchella species. Among seven tested chemicals (ampicillin, azithromycin, kanamycin, rifampicin, roxithromycin, acyclovir hydrochloride, and ribavirin), only ribavirin significantly inhibited MiEV1 replication. Successive ribavirin treatments eliminated MiEV1 from a representative M. sextelata strain (M11), with the virus-free status remaining stable after multiple subcultures on drug-free medium. Elimination of MiEV1 also significantly enhanced mycelial growth rate and biomass, and led to enhanced mycelial pigmentation, suggesting a potential negative impact of MiEV1 on vegetative growth and pigmentation in M. sextelata. These findings demonstrate that MiEV1 is prevalent in cultivated Morchella and that ribavirin provides an effective strategy for generating stable virus-free strains, offering opportunities for mechanistic studies and industrial cultivation.

真菌病毒破坏羊肚菌的培养。我们对24株培养菌株（8 M.）进行了5种分枝病毒的检测，分别为：羊肚菌内膜病毒1型[MiEV1]、羊肚菌内膜病毒2型[MiEV2]、羊肚菌内膜病毒3型[MiEV3]、羊肚菌镰刀病毒1型[MeFV1]和羊肚菌镰刀病毒2型[MeFV2]。importuna 5 M。septimelata和11 M。在中国11个省份收集的六角星（sextelata），并评估候选抗病毒药物。仅检测到MiEV1，在所有3种羊肚菌中发病率高达79.2% %（19/24）。在7种化学物质（氨苄西林、阿奇霉素、卡那霉素、利福平、罗红霉素、盐酸阿昔洛韦和利巴韦林）中，只有利巴韦林能显著抑制MiEV1的复制。连续的利巴韦林治疗消除了具有代表性的六角分枝杆菌菌株（M11）中的MiEV1，在无药培养基上多次传代培养后，其无病毒状态保持稳定。消除MiEV1还能显著提高菌丝生长速率和生物量，并导致菌丝色素沉着增强，表明MiEV1对六爪草的营养生长和色素沉着有潜在的负面影响。这些发现表明，MiEV1在培养的羊肚菌中普遍存在，利巴韦林为产生稳定的无病毒菌株提供了有效的策略，为机理研究和工业化培养提供了机会。

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引用次数: 0

Corrigendum to "Targeted enrichment of elephant endotheliotropic herpesvirus for complete genome sequencing in elephants" [J. Virol. Methods 338 (2025), 115230]. 大象嗜内皮疱疹病毒全基因组测序的靶向富集[J]。性研究。方法338(2025),115230]。

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2026-01-09 DOI: 10.1016/j.jviromet.2026.115348

Kirtika Sharma, Kg Sai Balaji, Gaurav Kumar Sharma, Abhijit M Pawde, Sonalika Mahajan, Ravikant Agrawal, Pracheta Janmeda, Karikalan Mathesh

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 : 2026-01-06 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

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 : 2026-01-05 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

Corrigendum to “An alternative model for HEV infection in the HepaRG cell line” [J. Virol. Methods 340 (2026) 115307] “HepaRG细胞系中HEV感染的另一种模型”的更正[J]。性研究。方法340(2026)115307。

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2025-12-24 DOI: 10.1016/j.jviromet.2025.115334

Stacy Gellenoncourt , Marie Pellerin , Aïlona Marcadet-Hauss , Roxanne Fouillé , Michel Rivoire , Guillaume Passot , Julie Lucifora , David Durantel , Nicole Pavio , Virginie Doceul

引用次数: 0

Evaluation of dried blood spot testing for serological monitoring of epizootic and zoonotic pathogens in domestic pigs 家猪兽疫和人畜共患病原体血清学监测中干血斑点试验的评价。

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2025-12-24 DOI: 10.1016/j.jviromet.2025.115336

Ranja Steinhauer , Eric Kübler , Stefan Gaugler , Cornel Fraefel , Julia Lechmann

Dried blood spots (DBS) constitute a stable, cost-efficient sampling matrix that can be collected in a minimally invasive manner. Although widely adopted in human medicine, their use in veterinary diagnostics remains limited. This study aimed to establish and validate DBS elution protocols for use in commercial ELISAs to detect antibodies against Hepatitis E virus (HEV), African swine fever virus (ASFV) and Aujeszky’s disease virus (ADV) in domestic pigs. DBS were prepared from EDTA blood, dried serum spots (DSS) from serum. Additional DBS samples were prepared after spiking blood from healthy pigs with antibodies. Various parameters were evaluated to establish the final elution protocols, i.e. number of disks, type and volume of elution buffer, incubation time of the disks in elution buffer, and volume of eluate used for detection. Once the final protocols were in place, for each pathogen 52 DBS were tested in three independent runs. The diagnostic performance was evaluated by comparing the ELISA results of DBS eluates with the corresponding serum or plasma samples. For HEV, only one out of 52 DBS samples qualitatively did not match the plasma result in any of the three runs. For ASFV, all 52 DBS samples matched the qualitative results of the corresponding liquid samples. For ADV, two samples yielded false negative results in all three runs. The results suggest that DBS represent a practical and reliable alternative to liquid blood samples for antibody detection in pigs. Further validation with field samples and large-scale testing is needed.

干血斑（DBS）构成了一种稳定、成本效益高的采样基质，可以以微创的方式采集。虽然在人类医学中被广泛采用，但它们在兽医诊断中的应用仍然有限。本研究旨在建立和验证DBS洗脱方案，用于商用elisa检测家猪戊型肝炎病毒（HEV）、非洲猪瘟病毒（ASFV）和奥杰斯基病病毒（ADV）的抗体。EDTA血制备DBS，血清干斑（DSS）制备DBS。在健康猪的血液中加入抗体后，制备了额外的DBS样本。评估各种参数以确定最终洗脱方案，包括：圆盘数、洗脱缓冲液的类型和体积、圆盘在洗脱缓冲液中的孵育时间、用于检测的洗脱液体积。一旦最终方案到位，每种病原体52个DBS在三个独立的运行中进行测试。通过比较DBS洗脱液的ELISA结果与相应的血清或血浆样品来评估诊断性能。对于HEV， 52个DBS样本中只有一个在三次运行中与血浆结果定性不匹配。对于ASFV， 52份DBS样品均与相应液体样品的定性结果相匹配。对于ADV，两个样本在所有三次运行中都产生了假阴性结果。结果表明，DBS是一种实用可靠的猪抗体检测方法，可替代液体血液样品。需要用现场样品和大规模试验进一步验证。

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引用次数: 0

Development of sensitive and specific indirect ELISA tests for the detection of antibodies against bovine leukemia virus in serum and milk samples 血清和牛奶中抗牛白血病病毒抗体的间接ELISA检测方法的建立。

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2025-12-23 DOI: 10.1016/j.jviromet.2025.115335

Andrés Addiego , Federico Carrión , Natalia Olivero-Deibe , Martín Fló , Florencia Rammauro , Natalia Ibañez , Caroline da Silva Silveira , Franklin Riet-Correa , Lorena Tomé-Poderti , Otto Pritsch , Sergio Bianchi

Bovine leukemia virus (BLV) is the causative agent of enzootic bovine leukosis (EBL), a disease characterized by lymphoproliferation and B-cell tumors. There is currently no available treatment or vaccine, making it a global animal health challenge and resulting in severe economic losses. The World Organization for Animal Health (WOAH) advocates for serological diagnostic tools and disease control programs to eradicate BLV. One major diagnostic technique recognized by the WOAH is the enzyme-linked immunosorbent assay (ELISA). We developed and optimized three indirect ELISAs for detecting anti-BLV antibodies in serum and milk. These assays utilize viral Env ectodomain and p24 recombinant proteins expressed in Drosophila melanogaster S2 cells and Escherichia coli systems, respectively. We compared their performance with a widely used commercial ELISA kit for completeness, which detects antibodies against BLV gp51 protein. Our immunoassay using recombinant p24 protein (anti-p24-S ELISA) showed comparable strength to the reference method (kappa index: 84.4 %, in 952 samples evaluated). Results for the anti-ectoEnv-S ELISA in serum samples closely matched the reference kit, with higher concordance than the anti-p24-S ELISA (kappa index: 94.9 %, in 1781 samples evaluated). Similarly, our in-house anti-ectoEnv-M ELISA in milk samples exhibited high concordance with both the commercial reference kit and the anti-ectoEnv-S (kappa index between the three tests: 95.6 %, in 479 blood and milk paired samples). Thus, we have developed, optimized, and validated three indirect in-house ELISAs for detecting anti-BLV antibodies in cattle, demonstrating good sensitivity and specificity values, using two different recombinant viral proteins.

牛白血病病毒（BLV）是地方性牛白血病（EBL）的病原体，EBL是一种以淋巴细胞增殖和b细胞肿瘤为特征的疾病。目前没有可用的治疗方法或疫苗，使其成为全球动物卫生挑战，并造成严重的经济损失。世界动物卫生组织（WOAH）提倡使用血清学诊断工具和疾病控制计划来根除BLV。世界卫生组织认可的一种主要诊断技术是酶联免疫吸附试验（ELISA）。我们开发并优化了3种间接elisa检测血清和乳汁中的抗blv抗体。这些实验分别利用在果蝇S2细胞和大肠杆菌系统中表达的病毒Env外结构域和p24重组蛋白。我们将其性能与广泛使用的商用ELISA试剂盒进行了比较，该试剂盒检测BLV gp51蛋白的抗体。我们使用重组p24蛋白（抗p24- s ELISA）进行免疫分析，其强度与参考方法相当（在评估的952份样品中kappa指数为84.4%）。血清样品中抗ectoenv - s酶联免疫吸附试验结果与参考试剂盒的一致性较好，且高于抗p24- s酶联免疫吸附试验（kappa指数为94.9%，共1781份）。同样，我们在牛奶样品中的抗ectoenv - m ELISA与商业参考试剂盒和抗ectoenv - s均表现出高度的一致性（在479份血液和牛奶配对样品中，三种检测的kappa指数为95.6%）。因此，我们利用两种不同的重组病毒蛋白，开发、优化并验证了三种用于检测牛体内抗blv抗体的间接elisa，显示出良好的灵敏度和特异性。

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引用次数: 0

Rapid establishment and validation of a PMAxx-RT-qPCR method for infectious titer detection of freeze-dried live attenuated hepatitis A vaccine (H2 Strain) 冻干甲型肝炎减毒活疫苗H2株感染效价的pmax - rt - qpcr快速检测方法的建立与验证

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2025-12-23 DOI: 10.1016/j.jviromet.2025.115333

Yongrong Wang , You Gao , Wei Wang , Qin Liu , Yadong Li , Haiyuan Zeng , Lifei Chen , Chen Cheng , Fan Wu

The infectious titer of hepatitis A virus (HAV) is a critical quality parameter for evaluating potency of freeze-dried live attenuated hepatitis A (HepA-L-fd) vaccines. In this study, we developed a rapid PMAxx-RT-qPCR for detecting the infectious titer of HepA-L-fd vaccines. This method enables virus titer detection to be completed within a day. Systematic optimization of experimental parameters yielded a validated methodology demonstrating excellent precision (coefficient of variation, CV < 1 %), accuracy (recovery rate 107.6 ± 3.7 %), and a linear range of 6.23–7.73 log₁₀CCID₅₀/mL. Comparing with the measurements from the traditional method, results showed no significant differences among the six batches of vaccine finished products (P = 0.7452). This optimized PMAxx-RT-qPCR assay provides a rapid and reliable analytical means for the infectious titer assay in HepA-L-fd vaccines.

甲型肝炎病毒（HAV）的感染效价是评价冻干甲型肝炎减毒活疫苗效价的重要质量参数。在这项研究中，我们建立了一种快速检测HepA-L-fd疫苗感染滴度的pmax - rt - qpcr。此方法可在一天内完成病毒滴度检测。对实验参数进行系统优化，得到精密度高（变异系数CV < 1%）、准确度高（回收率107.6±3.7%），线性范围为6.23 ~ 7.73 log10CCID50/mL。与传统方法比较，6批疫苗成品的测定结果无显著差异（P = 0.7452）。优化后的pmax - rt - qpcr检测方法为检测HepA-L-fd疫苗的感染滴度提供了一种快速可靠的分析方法。

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引用次数: 0

Application of digital PCR and CRISPR/Cas13a-based fluorescent assay for accurate and on-site detection of cotton leafroll dwarf virus 数字PCR和基于CRISPR/ cas13的荧光技术在棉花卷叶矮缩病毒准确和现场检测中的应用

IF 1.6 4区医学 Q3 BIOCHEMICAL RESEARCH METHODS

Journal of virological methods

Pub Date : 2025-12-19 DOI: 10.1016/j.jviromet.2025.115332

Vijay Sheri , Pankaj K. Verma , SaiKrishna Lekkala, Madhusudhana R. Janga

Cotton leafroll dwarf virus (CLRDV) is an emerging viral pathogen posing a significant threat to cotton production in the United States. Early and accurate detection is critical for effective disease surveillance and management. Although traditional reverse transcription PCR (RT-PCR) is commonly employed for CLRDV diagnosis, it suffers from limitations in sensitivity, quantification accuracy, and involves labor-intensive workflows. In this study, we evaluated two advanced molecular diagnostic approaches for detecting CLRDV, digital PCR (dPCR) and CRISPR/Cas13a-based fluorescent assay. Symptomatic cotton leaf samples from Lubbock and Brownfield, Texas, were screened and confirmed positive by RT-PCR. Digital PCR analysis enabled absolute quantification of viral load, revealing significantly higher titers in Brownfield (F2) samples and offered improved sensitivity over RT-PCR, particularly in samples with low viral loads. However, dPCR is resource-intensive and requires specialized instrumentation. To address the need for rapid, field-deployable diagnostics, we developed a CRISPR/Cas13a-based assay targeting the conserved ORF3, ORF2, and ORF3a regions of the CLRDV genome. Adapted from the SHERLOCK platform, this fluorescence-based assay uses collateral cleavage activity of Cas13a to enable highly specific visual detection. While the assay successfully enabled direct detection from crude leaf extracts without RNA purification, the sensitivity analysis was conducted using purified, in vitro transcribed RNA. Fluorescence signals were reliably observed with as few as 50 RNA copies, defining the assay’s practical limit of detection. While dPCR is optimal for quantitative laboratory analysis, the CRISPR/Cas13a-based assay offers a rapid, sensitive, and cost-effective tool for field-level detection. Together, these complementary tools enhance CLRDV surveillance and management in cotton.

棉花卷叶矮病毒（CLRDV）是一种新兴的病毒性病原体，对美国棉花生产构成重大威胁。早期和准确的检测对于有效的疾病监测和管理至关重要。虽然传统的逆转录PCR （RT-PCR）通常用于CLRDV的诊断，但它在灵敏度、定量准确性方面存在局限性，并且涉及劳动密集型的工作流程。在这项研究中，我们评估了检测CLRDV的两种先进的分子诊断方法，数字PCR （dPCR）和基于CRISPR/ cas13的荧光检测。对来自德克萨斯州Lubbock和Brownfield的有症状棉花叶片样本进行筛选，并通过RT-PCR确认为阳性。数字PCR分析实现了病毒载量的绝对定量，在Brownfield （F2）样品中显示出明显更高的滴度，并提供了比RT-PCR更高的灵敏度，特别是在低病毒载量的样品中。然而，dPCR是资源密集型的，需要专门的仪器。为了满足快速、可现场部署诊断的需求，我们开发了一种基于CRISPR/ cas13的检测方法，针对CLRDV基因组的保守ORF3、ORF2和ORF3a区域。这种基于荧光的检测方法改编自SHERLOCK平台，利用Cas13a的侧切活性来实现高度特异性的视觉检测。虽然该试验成功地从粗叶提取物中直接检测而无需纯化RNA，但使用纯化的体外转录RNA进行敏感性分析。荧光信号被可靠地观察到，只有50个RNA拷贝，确定了该分析的实际检测极限。虽然dPCR是定量实验室分析的最佳选择，但基于CRISPR/ cas13的分析为现场检测提供了快速、敏感和经济高效的工具。这些互补工具共同加强了棉花中CLRDV的监测和管理。

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引用次数: 0