A Multiplex TaqMan-MGB qPCR Assay for Rapid and Accurate Identification of Four Waterfowl Parvoviruses (cGPV, MDPV, MDGPV, and SBDSV)

IF 3 2区农林科学 Q2 INFECTIOUS DISEASES

Transboundary and Emerging Diseases Pub Date : 2025-09-28 DOI:10.1155/tbed/9999490

Min Zheng, Dandan Jiang, Shifeng Xiao, Shao Wang, Xiaoxia Cheng, Xiaoli Zhu, Xiuqin Chen, Meiqing Huang, Shaoying Chen, Shilong Chen

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引用次数: 0

Abstract

Waterfowl parvoviruses (WPVs), including classical goose parvovirus (cGPV), Muscovy duck parvovirus (MDPV), Muscovy duck-origin goose parvovirus (MDGPV), and short beak and dwarfism syndrome virus (SBDSV), are significant pathogens that affect waterfowl flocks worldwide. Due to their high genetic similarity and frequent coinfections, rapid and accurate differentiation of these viruses remains challenging. In this study, we developed a multiplex TaqMan-minor groove binder (MGB) real-time PCR assay for the simultaneous detection and differentiation of cGPV, MDPV, MDGPV, and SBDSV. Specific primers and TaqMan-MGB probes were designed based on sequence alignments of the VP gene. This assay exhibited high specificity, with no cross-reactivity to other main waterfowl viruses. The detection limits of this assay were 10² copies/μL for cGPV, 10¹ copies/μL for MDPV, 10² copies/μL for MDGPV, and 10³ copies/μL for SBDSV, respectively. The standard curves exhibited strong linearity (R²≥0.995) and high amplification efficiency (89%–108%), with intra- and interassay coefficients of variation below 2.0%, indicating high repeatability and stability. Clinical testing of 337 clinical samples suspected of WPV infection demonstrated that the developed assay outperformed conventional PCR, achieving higher overall detection rates (58% vs 54%) and enhanced identification of coinfections. Epidemiological analysis revealed MDGPV as the predominant circulating strain in Muscovy ducks, with 27 samples identified as coinfected with both MDGPV and MDPV, while SBDSV showed higher prevalence in mule ducks and Pekin ducks. Notably, MDGPV was detected for the first time in goslings. These findings provide clear evidence of ongoing host restriction and potential cross-species transmission of WPVs among duck flocks. In conclusion, the multiplex TaqMan-MGB quantitative PCR (qPCR) assay developed in this study provides a rapid, sensitive, and reliable tool for the simultaneous detection and differentiation of cGPV, MDPV, MDGPV, and SBDSV. Its application is expected to enhance disease surveillance, facilitate outbreak control, and contribute to more effective control of waterfowl parvoviral diseases.

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快速准确鉴定水禽细小病毒（cGPV、MDPV、MDGPV和SBDSV）的多重TaqMan-MGB qPCR方法

水禽细小病毒（wpv）包括鹅细小病毒（cGPV）、幕鸭细小病毒（MDPV）、幕鸭源鹅细小病毒（MDGPV）和短喙与侏儒症综合征病毒（SBDSV），是影响全世界水禽群的重要病原体。由于它们的高度遗传相似性和频繁的共感染，快速和准确地区分这些病毒仍然具有挑战性。在这项研究中，我们建立了多重TaqMan-minor groove binder （MGB）实时PCR方法，用于同时检测和分化cGPV、MDPV、MDGPV和SBDSV。根据VP基因序列比对设计特异性引物和TaqMan-MGB探针。该方法特异性高，与其他主要水禽病毒无交叉反应。检测限分别为cGPV 102拷贝/μL、MDPV 101拷贝/μL、MDGPV 102拷贝/μL、SBDSV 10³拷贝/μL。标准曲线线性强（R2≥0.995），扩增效率高（89% ~ 108%），内、间变异系数均小于2.0%，重复性和稳定性好。对337份疑似WPV感染的临床样本进行的临床测试表明，所开发的检测方法优于传统PCR，实现了更高的总体检出率（58%对54%），并增强了对合并感染的识别。流行病学分析显示，MDGPV是莫斯科鸭的主要流行毒株，有27份样本同时感染MDGPV和MDPV，而SBDSV在骡鸭和北京鸭中的流行率较高。值得注意的是，MDGPV首次在雏鹅中检测到。这些发现提供了明确的证据，表明野生脊灰病毒在鸭群中存在持续的宿主限制和潜在的跨种传播。总之，本研究建立的多重TaqMan-MGB定量PCR （qPCR）方法为同时检测和区分cGPV、MDPV、MDGPV和SBDSV提供了一种快速、灵敏、可靠的工具。它的应用有望加强疾病监测，促进疫情控制，并有助于更有效地控制水禽细小病毒疾病。

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来源期刊

Transboundary and Emerging Diseases 农林科学-传染病学

CiteScore

8.90

自引率

9.30%

发文量

350

审稿时长

1 months

期刊介绍： Transboundary and Emerging Diseases brings together in one place the latest research on infectious diseases considered to hold the greatest economic threat to animals and humans worldwide. The journal provides a venue for global research on their diagnosis, prevention and management, and for papers on public health, pathogenesis, epidemiology, statistical modeling, diagnostics, biosecurity issues, genomics, vaccine development and rapid communication of new outbreaks. Papers should include timely research approaches using state-of-the-art technologies. The editors encourage papers adopting a science-based approach on socio-economic and environmental factors influencing the management of the bio-security threat posed by these diseases, including risk analysis and disease spread modeling. Preference will be given to communications focusing on novel science-based approaches to controlling transboundary and emerging diseases. The following topics are generally considered out-of-scope, but decisions are made on a case-by-case basis (for example, studies on cryptic wildlife populations, and those on potential species extinctions): Pathogen discovery: a common pathogen newly recognised in a specific country, or a new pathogen or genetic sequence for which there is little context about — or insights regarding — its emergence or spread. Prevalence estimation surveys and risk factor studies based on survey (rather than longitudinal) methodology, except when such studies are unique. Surveys of knowledge, attitudes and practices are within scope. Diagnostic test development if not accompanied by robust sensitivity and specificity estimation from field studies. Studies focused only on laboratory methods in which relevance to disease emergence and spread is not obvious or can not be inferred (“pure research” type studies). Narrative literature reviews which do not generate new knowledge. Systematic and scoping reviews, and meta-analyses are within scope.