Yehia Nahed, Eldemery Fatma, Arafa Abdel-Satar, Abd El Wahed Ahmed, El Sanousi Ahmed, Weidmann Manfred, Shalaby Mohamed
National Laboratory for Quality Control on Poultry Production, Animal Health Research Institute, Agriculture Research Center, Dokki, Giza 12618, Egypt.
Department of Hygiene and Zoonoses, Faculty of Veterinary Medicine, Mansoura University, Mansoura 35516, Egypt.
Vet Sci. 2021 Jul 16;8(7):134. doi: 10.3390/vetsci8070134.
The H9N2 subtype of avian influenza A virus (aIAV) is circulating among birds worldwide, leading to severe economic losses. H9N2 cocirculation with other highly pathogenic aIAVs has the potential to contribute to the rise of new strains with pandemic potential. Therefore, rapid detection of H9 aIAVs infection is crucial to control virus spread. A qualitative reverse transcription recombinase polymerase amplification (RT-RPA) assay for the detection of aIAV subtype H9N2 was developed. All results were compared to the gold standard (real-time reverse transcription polymerase chain reaction (RT-PCR)). The RT-RPA assay was designed to detect the hemagglutinin (HA) gene of H9N2 by testing three pairs of primers and a probe. A serial concentration between 10 and 10 EID (50% embryo infective dose)/mL was applied to calculate the analytical sensitivity. The H9 RT-RPA assay was highly sensitive as the lowest concentration point of a standard range at one EID/mL was detected after 5 to 8 min. The H9N2 RT-RPA assay was highly specific as nucleic acid extracted from H9 negative samples and from other avian pathogens were not cross detected. The diagnostic sensitivity when testing clinical samples was 100% for RT-RPA and RT-PCR. In conclusion, H9N2 RT-RPA is a rapid sensitive and specific assay that easily operable in a portable device for field diagnosis of aIAV H9N2.
甲型禽流感病毒（aIAV）的H9N2亚型在全球鸟类中传播，导致严重的经济损失。H9N2与其他高致病性aIAVs共同传播有可能促使具有大流行潜力的新毒株出现。因此，快速检测H9 aIAVs感染对于控制病毒传播至关重要。开发了一种用于检测aIAV亚型H9N2的定性逆转录重组酶聚合酶扩增（RT-RPA）检测方法。所有结果均与金标准（实时逆转录聚合酶链反应（RT-PCR））进行比较。RT-RPA检测方法通过测试三对引物和一个探针来检测H9N2的血凝素（HA）基因。应用10至10 EID（50%胚胎感染剂量）/mL之间的系列浓度来计算分析灵敏度。H9 RT-RPA检测方法高度灵敏，在5至8分钟后检测到标准范围的最低浓度点为1 EID/mL。H9N2 RT-RPA检测方法高度特异，未交叉检测到从H9阴性样本和其他禽病原体中提取的核酸。检测临床样本时，RT-RPA和RT-PCR的诊断灵敏度均为100%。总之，H9N2 RT-RPA是一种快速、灵敏且特异的检测方法，可轻松在便携式设备中操作，用于现场诊断aIAV H9N2。
