Advancing multiplex diagnostics: A novel framework for simultaneous detection of diverse pathogens using respiratory RNA viruses as a model system

IF 1.6 4区医学 Q4 BIOCHEMICAL RESEARCH METHODS

Journal of immunological methods Pub Date : 2025-04-04 DOI:10.1016/j.jim.2025.113857

M. Caputo , S. Ginart , L. Garrigos , D. Corach , F. Remes Lenicov , A. Sala

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引用次数: 0

Abstract

Numerous infectious pathologies share clinical presentation but require different management. The search for multiple pathogens in a single test could improve the resolution of these cases. For example respiratory infections, caused either by viral, bacterial or fungal agents. Rapid differential detection of common respiratory RNA viruses could be benefit by such a strategy.

We present a diagnostic method based on real-time quantitative PCR (qPCR) coupled with high-resolution melt (HRM) analysis. While applicable to diverse pathogens, this study focuses on detecting three respiratory RNA viruses: influenza A (H1N1), respiratory syncytial virus (RSV), and SARS-CoV-2.

To develop a rapid, specific, and cost-effective system, we designed a multiplex assay using SYTO 9 fluorescent dye and pathogen-specific primers. Post-amplification HRM analysis generated melt curves, with first-derivative plots enabling discrimination of the three viruses.

Samples tested in quintuplicate (10¹–10³ copies/μL) yielded distinct melt peaks. Mean melting temperatures (average Tm ± SD) were 83.64 ± 0.08 °C (SARS-CoV-2), 80.45 ± 0.07 °C (influenza H1N1), and 75.99 ± 0.07 °C (RSV). The system robustly differentiated all three viruses (100 % accuracy). Preliminary validation with clinical samples showed strong agreement with a commercial kit: κ = 0.87 (95 % CI: 0.61–1.00) for SARS-CoV-2 and κ = 1.00 (95 % CI: 0.74–1.00) for RSV.

Therefore, the designed method represents a rapid and cost-effective alternative for differential diagnosis in a single reaction tube.

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推进多重诊断：一个使用呼吸道RNA病毒作为模型系统同时检测多种病原体的新框架。

许多传染性病理具有相同的临床表现，但需要不同的处理。在一次测试中寻找多种病原体可以提高这些病例的分辨率。例如，由病毒、细菌或真菌引起的呼吸道感染。这种策略可能有利于对常见呼吸道RNA病毒的快速鉴别检测。我们提出了一种基于实时定量PCR （qPCR）和高分辨率熔体（HRM）分析的诊断方法。虽然适用于多种病原体，但本研究主要检测三种呼吸道RNA病毒：甲型H1N1流感病毒、呼吸道合胞病毒（RSV）和SARS-CoV-2。为了开发一种快速、特异且具有成本效益的系统，我们设计了一种使用SYTO 9荧光染料和病原体特异性引物的多重检测方法。扩增后HRM分析生成熔体曲线，一阶导数图能够区分三种病毒。样品在5个副本（101-103拷贝/μL）中得到明显的熔体峰。意味着融化温度(平均Tm ± SD)是83.64 ±0.08  °C (SARS-CoV-2), 80.45 ±0.07  °C(甲型H1N1流感)和75.99 ±0.07  °C (RSV)。该系统稳健地区分了所有三种病毒（100% %准确率）。临床样品的初步验证显示与商业试剂盒高度一致：SARS-CoV-2的κ = 0.87(95 % CI: 0.61-1.00)， RSV的κ = 1.00（95 % CI: 0.74-1.00）。因此，所设计的方法代表了在单个反应管中进行鉴别诊断的快速和具有成本效益的替代方法。

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

Journal of immunological methods 医学-免疫学

CiteScore

4.10

自引率

0.00%

发文量

120

审稿时长

3 months

期刊介绍： The Journal of Immunological Methods is devoted to covering techniques for: (1) Quantitating and detecting antibodies and/or antigens. (2) Purifying immunoglobulins, lymphokines and other molecules of the immune system. (3) Isolating antigens and other substances important in immunological processes. (4) Labelling antigens and antibodies. (5) Localizing antigens and/or antibodies in tissues and cells. (6) Detecting, and fractionating immunocompetent cells. (7) Assaying for cellular immunity. (8) Documenting cell-cell interactions. (9) Initiating immunity and unresponsiveness. (10) Transplanting tissues. (11) Studying items closely related to immunity such as complement, reticuloendothelial system and others. (12) Molecular techniques for studying immune cells and their receptors. (13) Imaging of the immune system. (14) Methods for production or their fragments in eukaryotic and prokaryotic cells. In addition the journal will publish articles on novel methods for analysing the organization, structure and expression of genes for immunologically important molecules such as immunoglobulins, T cell receptors and accessory molecules involved in antigen recognition, processing and presentation. Submitted full length manuscripts should describe new methods of broad applicability to immunology and not simply the application of an established method to a particular substance - although papers describing such applications may be considered for publication as a short Technical Note. Review articles will also be published by the Journal of Immunological Methods. In general these manuscripts are by solicitation however anyone interested in submitting a review can contact the Reviews Editor and provide an outline of the proposed review.