Abstract
Recent studies have demonstrated that at least eight subtypes of avian influenza virus (AIV) can infect humans, including H1, H2, H3, H5, H6, H7, H9 and H10. A GeXP analyser-based multiplex reverse transcription (RT)-PCR (GeXP-multiplex RT-PCR) assay was developed in our recent studies to simultaneously detect these eight AIV subtypes using the haemagglutinin (HA) gene. The assay consists of chimeric primer-based PCR amplification with fluorescent labelling and capillary electrophoresis separation. RNA was extracted from chick embryo allantoic fluid or liquid cultures of viral isolates. In addition, RNA synthesised via in vitro transcription was used to determine the specificity and sensitivity of the assay. After selecting the primer pairs, their concentrations and GeXP-multiplex RT-PCR conditions were optimised. The established GeXP-multiplex RT-PCR assay can detect as few as 100 copies of premixed RNA templates. In the present study, 120 clinical specimens collected from domestic poultry at live bird markets and from wild birds were used to evaluate the performance of the assay. The GeXP-multiplex RT-PCR assay specificity was the same as that of conventional RT-PCR. Thus, the GeXP-multiplex RT-PCR assay is a rapid and relatively high-throughput method for detecting and identifying eight AIV subtypes that may infect humans.
Highlights
Influenza A viruses are important human and animal pathogens affecting human health, causing severe animal diseases and death
Molecular biological diagnostic methods based on PCR technology, such as conventional PCR, reverse transcription PCR (RT-PCR), real-time RT-PCR (RRT-PCR) and quantitative RRT-PCR have been widely used for avian influenza virus (AIV) detection and genotyping[16,17,18,19]
The target matrix (M) gene was amplified from all AIV subtypes where each specific primer pair could not cross-amplify other HA AIV subtypes but could amplify only the corresponding target gene (Table S1)
Summary
Influenza A viruses are important human and animal pathogens affecting human health, causing severe animal diseases and death. Studies have shown that AIV subtypes H1, H2, H3, H5, H7, H9 and H10 can directly infect humans and could be potentially lethal pathogens that cause human influenza pandemics. In 2017, H7N9 AIV mutated into a strain that is highly pathogenic against chickens and caused hundreds of cases of human infections in China[11,12,13,14]. Several human and animal pathogens, including those causing hand, foot and mouth disease, 16 human respiratory viral types or subtypes, and 11 human papilloma viruses have been successfully and rapidly detected and identified using the GeXP analyser[23,24,25]. This report describes our recently developed multiplex RT-PCR assay using a GeXP analyser (GeXP-multiplex RT-PCR) to simultaneously detect eight AIV subtypes that can infect humans
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