Coopersmith Matthew, Dijkman Remco, Bartlett Maggie L, Currie Richard, Schuurman Sander, Wit Sjaak de
Alveo Technologies, Inc., Alameda, CA 94501, USA.
Department of R&D, Royal GD, 7418 EZ Deventer, The Netherlands.
Microorganisms. 2025 May 8;13(5):1090. doi: 10.3390/microorganisms13051090.
Avian influenza A viruses (AIV) significantly impact both animal and human health. Reliable diagnostics are crucial for controlling AIV, including the highly pathogenic strains like H5Nx. In this study, we developed and validated the on-site Alveo Sense Poultry Avian Influenza Tests to rapidly detect the AIV M-gene and subtypes H5, H7, and H9 in unprocessed samples using reverse-transcription loop-mediated isothermal amplification (RT-LAMP) and impedance-based measurements. The Alveo Sense tests, using single-use microfluidic cartridges, deliver results within 45 min. Each cartridge includes assays for the AIV M gene and specific H5 and H7 or H9 subtypes, with internal process controls. The laboratory validation involved specificity, limit of detection (LoD), diagnostic sensitivity, reproducibility, and robustness tests using various AIV strains, other avian pathogens, and field samples. The assays showed 100% specificity for AIV subtypes without cross-reactivity with non-AIV pathogens. The LoD95 for H5, H7, and H9 ranged between RT-PCR Ct values of 29-33 in both cloacal and oropharyngeal samples and were able to detect avian influenza virus in both spiked samples and field samples. Reproducibility and repeatability studies showed perfect agreement across operators and laboratories and remained stable and accurate under different pre-analytical conditions. The Alveo Sense tests offer rapid, accurate, and reliable on-site diagnostics for AIV subtypes H5, H7, and H9 on samples from fresh dead and sick birds, valuable for early flock-level detection and outbreak control. Further field validation will improve the understanding of their diagnostic performance across various avian species.
甲型禽流感病毒（AIV）对动物和人类健康都有重大影响。可靠的诊断对于控制AIV至关重要，包括H5Nx等高致病性毒株。在本研究中，我们开发并验证了现场Alveo Sense家禽禽流感检测方法，该方法使用逆转录环介导等温扩增（RT-LAMP）和基于阻抗的测量技术，能够在未处理的样本中快速检测AIV的M基因以及H5、H7和H9亚型。Alveo Sense检测使用一次性微流控芯片，可在45分钟内得出结果。每个芯片都包括针对AIV M基因以及特定H5和H7或H9亚型的检测，并设有内部过程控制。实验室验证包括特异性、检测限（LoD）、诊断敏感性、重现性和稳健性测试，使用了各种AIV毒株、其他禽类病原体和现场样本。这些检测方法对AIV亚型显示出100%的特异性，与非AIV病原体无交叉反应。H5、H7和H9的LoD95在泄殖腔和口咽样本中的RT-PCR Ct值范围为29 - 33，能够在加标样本和现场样本中检测到禽流感病毒。重现性和重复性研究表明，不同操作人员和实验室之间结果完全一致，并且在不同的分析前条件下保持稳定和准确。Alveo Sense检测方法可为来自新鲜死亡和患病禽类的样本提供快速、准确且可靠的H5、H7和H9亚型现场诊断，这对于早期禽群水平的检测和疫情控制具有重要价值。进一步的现场验证将有助于更好地了解其在各种禽类中的诊断性能。
