Highly pathogenic avian influenza H5N1 virus causes heavy losses in poultry farms worldwide. Molecular diagnostic techniques like RT-PCR and real-time RT-PCR are considered the gold standard for identification of H5 influenza viruses in clinical samples. These techniques are hampered by the need of well-equipped laboratories, large space requirement, and relatively long time-to-result. Recombinase polymerase amplification (RPA) assay represents an excellent alternative to PCR since it is more simple, rapid, economic, and portable. Reverse transcription RPA (RT-RPA) assay was recently developed for sensitive and specific detection of H5N1 virus in 6-10 min. To ensure the accuracy of the developed assay, two approaches for using a positive control were evaluated in this study. These approaches included: 1) all-in-one (internal positive control; IPC), 2) two-tubes-per-one-sample (external positive control; EPC). Sigma virus (SIGV) RNA and turkey mitochondrial DNA were tested as positive controls in both approaches. For all-in-one approach, both targets (H5 and IPC) were strongly inhibited. In contrast, very good amplification signals were obtained for the two types of EPC with no effect on the analytical sensitivity and specificity of H5 RT-RPA assay in two-tubes-per-one-sample approach. The performance of EPC-based H5 RT-RPA was further validated using 13 tracheal swabs. The results were compared to real-time RT-PCR and proved superior specificity in detecting H5N1 but not H5N8 viruses. Inclusion of EPC did not affect the aptitude of both assays in terms of sensitivity, specificity and reproducibility. In conclusion, the two-tubes-per-one-sample approach was more reliable to control the false negative results in H5 RT-RPA assay.

中文翻译：

---

评价使用阳性对照标准化禽流感H5逆转录重组酶聚合酶扩增试验的两种方法。

高致病性禽流感H5N1病毒在全世界的家禽养殖场造成了严重损失。诸如RT-PCR和实时RT-PCR的分子诊断技术被认为是鉴定临床样品中H5流感病毒的金标准。这些技术由于设备齐全的实验室的需要，较大的空间需求以及相对较长的开发时间而受到阻碍。重组酶聚合酶扩增（RPA）测定法是PCR的极佳替代方法，因为它更简单，快速，经济和便携。最近开发了逆转录RPA（RT-RPA）测定法，可在6-10分钟内灵敏而特异地检测H5N1病毒。为了确保开发的测定的准确性，本研究评估了两种使用阳性对照的方法。这些方法包括：1）多合一（内部阳性对照； IPC），2）每个样品两支试管（外部阳性对照； EPC）。两种方法均检测了Sigma病毒（SIGV）RNA和火鸡线粒体DNA作为阳性对照。对于多合一方法，两个目标（H5和IPC）均被强烈抑制。相比之下，对于两种类型的EPC，获得了非常好的扩增信号，而对两管一样品的H5 RT-RPA分析的灵敏度和特异性没有影响。使用13个气管拭子进一步验证了基于EPC的H5 RT-RPA的性能。将结果与实时RT-PCR进行比较，并证明了在检测H5N1病毒而非H5N8病毒方面具有优越的特异性。就敏感性，特异性和可重复性而言，加入EPC不会影响这两种测定的适用性。结论，