Abstract
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 106 and 100 EID50 (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 EID50/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.
Highlights
The Influenza A viruses (IAVs) genome is segmented into eight RNA segments: RNA polymerase basic subunit (PB)2, PB1, RNA polymerase acidic subunit (PA), hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), matrix (M)
We aimed to develop a probe-based reverse transcription recombinase polymerase amplification (RT-recombinase polymerase amplification (RPA)) and evaluated its performance for field detection of the HA gene of avian influenza H9N2
An efficient primer set is crucial for the optimization of the RT-RPA assay
Summary
Influenza A viruses (IAVs) are enveloped, single-stranded, negative-sense RNA viruses [1]. The IAV genome is segmented into eight RNA segments: RNA polymerase basic subunit (PB), PB1, RNA polymerase acidic subunit (PA), hemagglutinin (HA), nucleoprotein (NP), neuraminidase (NA), matrix (M) M2)), and non-structural (NS) segment [2]. Influenza A viruses are divided into subtypes based on HA and NA. 18 HA subtypes and 11 NA subtypes have been identified many different combinations of HA and NA exist [3]. H9N2 aIAVs continue to evolve and circulate in different bird species and poultry, and pose a public health concern [4]
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