Establishment of a multiplex qPCR assay for the detection of pathogens associated with bovine respiratory disease complex.

IF 2.6 2区农林科学 Q1 VETERINARY SCIENCES

Frontiers in Veterinary Science Pub Date : 2025-04-22 eCollection Date: 2025-01-01 DOI:10.3389/fvets.2025.1594488

Linghao Li, Qifeng Jiang, Siying Li, Xin Li, Shenghe Sun, Xiyi Wang, Chuangqi Sun, Kun Jia, Shoujun Li

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引用次数: 0

Abstract

Introduction: The bovine respiratory disease complex poses a significant threat to the cattle industry, necessitating a multifaceted approach to address its occurrence. The syndrome is caused by various pathogens such as bovine respiratory syncytial virus (BRSV), bovine parainfluenza virus type 3 (BPIV3), bovine viral diarrhea virus (BVDV), bovine adenovirus type 3 (BAV3), Mycoplasma bovis (Mb), and infectious bovine rhinotracheitis virus (IBRV). The confluence of these pathogens causes substantial economic losses to the cattle industry. Although preventive and control measures have been implemented, containment of bovine respiratory diseases continues to present a formidable challenge, highlighting the need for innovative diagnostic and intervention strategies.

Methods: In this study, we designed specific primers targeting six conserved pathogen genes (N of BRSV, M of BPIV3, 5'UTR of BVDV, Hexon of BAV3, oppF of Mb, and gB of IBRV). Subsequently, we established a multiplexed fluorescent real-time quantitative PCR (qPCR) assay for simultaneous detection of these pathogens.

Results: The developed method exhibited high specificity and sensitivity, with the lowest detection limits for plasmid DNA standards of BRSV, BPIV3, BVDV, BAV3, Mb, and IBRV being 70.1, 40.4, 15.1, 74.4, 69.6, and 4.99 copies/μL, respectively. The coefficients of variation determined by the assay established in this study were <4%, and the amplification efficiency was 93.84%-111.60%, which showed the reliability and stability of the method.

Discussion: The detection rates for BRSV, BPIV3, BVDV, BAV3, Mb, and IBRV were 7.59% (17/224), 11.61% (26/224), 8.04% (18/224), 22.32% (50/224), 27.23% (61/224), and 8.04% (18/224), respectively. All 224 cows were cases of natural disease. Fifty-six diseased cattle were infected with a mixture of two or more of the six pathogens at a mixed infection rate of 25% (56/224). Therefore, this study successfully developed a highly efficient, rapid, specific, and sensitive multiplex qPCR method to detect major pathogens associated with bovine respiratory diseases. This advancement is expected to significantly influence the future of the cattle industry and serve as a valuable reference for subsequent research in this field.

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建立一种多重qPCR方法检测与牛呼吸道疾病复合体相关的病原体。

牛呼吸道疾病对养牛业构成重大威胁，需要采取多方面的方法来解决其发生。该综合征由多种病原体引起，如牛呼吸道合胞病毒（BRSV）、牛副流感病毒3型（BPIV3）、牛病毒性腹泻病毒（BVDV）、牛腺病毒3型（BAV3）、牛支原体（Mb）和牛传染性鼻气管炎病毒（IBRV）。这些病原体的汇合给养牛业造成了巨大的经济损失。虽然已经实施了预防和控制措施，但遏制牛呼吸道疾病仍然是一项艰巨的挑战，这突出表明需要创新的诊断和干预战略。方法：针对BRSV的N基因、BPIV3的M基因、BVDV的5′utr基因、BAV3的Hexon基因、Mb的oppF基因和IBRV的gB基因设计特异性引物。随后，我们建立了一种多重荧光实时定量PCR （qPCR）方法来同时检测这些病原体。结果：该方法具有较高的特异性和灵敏度，对BRSV、BPIV3、BVDV、BAV3、Mb和IBRV的质粒DNA标准的最低检出限分别为70.1、40.4、15.1、74.4、69.6和4.99拷贝/μL。讨论：BRSV、BPIV3、BVDV、BAV3、Mb和IBRV的检出率分别为7.59%（17/224）、11.61%（26/224）、8.04%（18/224）、22.32%（50/224）、27.23%（61/224）和8.04%（18/224）。所有224头牛都是自然疾病。56头病牛被6种病原菌中的2种或2种以上混合感染，混合感染率为25%（56/224）。因此，本研究成功建立了一种高效、快速、特异、灵敏的多重qPCR检测牛呼吸道疾病相关主要病原体的方法。这一进展预计将对养牛业的未来产生重大影响，并为该领域的后续研究提供有价值的参考。

本文章由计算机程序翻译，如有差异，请以英文原文为准。

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来源期刊

Frontiers in Veterinary Science Veterinary-General Veterinary

CiteScore

4.80

自引率

9.40%

发文量

1870

审稿时长

14 weeks

期刊介绍： Frontiers in Veterinary Science is a global, peer-reviewed, Open Access journal that bridges animal and human health, brings a comparative approach to medical and surgical challenges, and advances innovative biotechnology and therapy. Veterinary research today is interdisciplinary, collaborative, and socially relevant, transforming how we understand and investigate animal health and disease. Fundamental research in emerging infectious diseases, predictive genomics, stem cell therapy, and translational modelling is grounded within the integrative social context of public and environmental health, wildlife conservation, novel biomarkers, societal well-being, and cutting-edge clinical practice and specialization. Frontiers in Veterinary Science brings a 21st-century approach—networked, collaborative, and Open Access—to communicate this progress and innovation to both the specialist and to the wider audience of readers in the field. Frontiers in Veterinary Science publishes articles on outstanding discoveries across a wide spectrum of translational, foundational, and clinical research. The journal''s mission is to bring all relevant veterinary sciences together on a single platform with the goal of improving animal and human health.