Auer Agathe, Panzarin Valentina, Monne Isabella, Crimaudo Marika, Angot Angelique, Gourlaouen Morgane, Lamien Charles E, Cattoli Giovanni
Emergency Prevention System for Animal Health (EMPRES-AH), Animal Health Service (NSAH), Food and Agriculture Organization of the United Nations (FAO-UN), Rome, Italy; Animal Production and Health Laboratory, Joint FAO/IAEA Centre for Nuclear Applications in Food and Agriculture, Department of Nuclear Sciences and Applications, International Atomic Energy Agency, Seibersdorf, Austria.
EU/WOAH/National Reference Laboratory for Avian Influenza and Newcastle Disease, FAO Reference Centre for Animal Influenza and Newcastle Disease, Division of Comparative Biomedical Sciences, Istituto Zooprofilattico Sperimentale delle Venezie (IZSVe), 35020 Legnaro, Italy.
J Virol Methods. 2023 Apr;314:114686. doi: 10.1016/j.jviromet.2023.114686. Epub 2023 Jan 31.
Global surveillance for Avian Influenza Virus (AIV) in birds is essential for assessing public and animal health risks and real-time polymerase chain reaction (RT-qPCR) is among the official methods recommended by the World Organisation for Animal Health (WOAH) to confirm the presence of the virus in laboratory specimens. Yet, in low-resource setting laboratories, the detection of AIV can be hampered by the need to maintain a cold chain for wet reagents. In such cases, alternatives should be ready to maximize surveillance capacities and mining of AIV. Therefore, we compared two lyophilized RT-qPCR reagents (1st - 5 × CAPITAL™ 1-Step qRT-PCR Probe Reagent, lyophilized kit, and 2nd - Qscript lyo 1-step-kit) to the WOAH recommended protocol by Nagy et al., 2020 using QuantiTect Probe RT-PCR-kit as wet reagent. The comparative study panel comprised 102 RNA samples from two AIV subtypes, i.e. H5 and H9 subtypes. Despite that the wet reagent exhibited the lowest limit of detection (LOD) compared to the two lyophilized reagents, the inter-assay agreement was substantial between the 1st lyophilized reagent and the comparator with 95.1% of shared positive results. Cohen's-kappa was fair between the 2nd lyophilized reagent and the comparator with 75.5% of shared positive results. Agreement using the statistical test Bland-Altman was good for samples with Cq-values < 25 for all reagents, revealing discrepancies when the viral load is low. This trend was especially evident while using the 2nd lyophilized reagent. Similar trends were obtained using the same lyophilized reagents but following the protocol by Heine et al., 2015 with AgPath-ID™ One-Step RT-PCR as a comparator, showing that Cq-values increase using lyophilized reagents but correlate strongly with the wet reagent. Further, inter-assay agreement between reagents improved when the protocol from Heine et al., 2015 was applied, suggesting a higher resilience to chemistry changes allowing easier reagents interchangeability.
对鸟类中的禽流感病毒（AIV）进行全球监测对于评估公共卫生和动物健康风险至关重要，实时聚合酶链反应（RT-qPCR）是世界动物卫生组织（WOAH）推荐的用于在实验室标本中确认病毒存在的官方方法之一。然而，在资源匮乏地区的实验室中，由于需要对湿试剂维持冷链，AIV的检测可能会受到阻碍。在这种情况下，应准备好替代方法以最大限度地提高监测能力和对AIV的检测。因此，我们将两种冻干的RT-qPCR试剂（第一种——5×CAPITAL™一步法qRT-PCR探针试剂，冻干试剂盒，第二种——Qscript lyo一步法试剂盒）与WOAH推荐的由Nagy等人在2020年提出的方案进行了比较，使用QuantiTect探针RT-PCR试剂盒作为湿试剂。比较研究小组包括来自两种AIV亚型（即H5和H9亚型）的102个RNA样本。尽管与两种冻干试剂相比，湿试剂的检测限最低，但第一种冻干试剂与对照之间的批间一致性很高，共享阳性结果的比例为95.1%。第二种冻干试剂与对照之间的Cohen's-kappa值一般，共享阳性结果的比例为75.5%。对于所有试剂，使用统计检验Bland-Altman法得出的一致性在Cq值<25的样本中良好，在病毒载量较低时显示出差异。使用第二种冻干试剂时，这种趋势尤为明显。使用相同的冻干试剂，但遵循Heine等人在2015年提出的方案，以AgPath-ID™一步法RT-PCR作为对照，也得到了类似的趋势，表明使用冻干试剂时Cq值会增加，但与湿试剂密切相关。此外，当应用Heine等人在2015年提出的方案时，试剂之间的批间一致性得到改善，这表明对化学变化的适应性更强，使得试剂更容易互换。
