title: Development of reverse transcription recombinase polymerase amplification assay for avian influenza H5N1 HA gene detection.
abstract: The 2006 outbreaks of H5N1 avian influenza in Egypt interrupted poultry production and caused staggering economic damage. In addition, H5N1 avian influenza viruses represent a significant threat to public health. Therefore, the rapid detection of H5 viruses is very important in order to control the disease. In this study, a qualitative reverse transcription recombinase polymerase amplification (RT-RPA) assay for the detection of hemagglutinin gene of H5 subtype influenza viruses was developed. The results were compared to the real-time reverse transcription polymerase chain reaction (RT-PCR). An in vitro transcribed RNA standard of 970 nucleotides of the hemagglutinin gene was developed and used to determine the assay sensitivity. The developed H5 RT-RPA assay was able to detect one RNA molecule within 7 min, while in real-time RT-PCR, at least 90 min was required. H5 RT-RPA assay did not detect nucleic acid extracted from H5 negative samples or from other pathogens producing respiratory manifestation in poultry. The clinical performance of the H5 RT-RPA assay was tested in 30 samples collected between 2014 and 2015; the sensitivity of H5 RT-RPA and real-time RT-PCR was 100%. In conclusion, H5 RT-RPA was faster than real-time RT-PCR and easily operable in a portable device. Moreover, it had an equivalent sensitivity and specificity.
authors.affiliation: National Laboratory for Veterinary Quality Control on Poultry Production, Animal Health Research Institute, Dokki, Giza 12618, Egypt. Electronic address: nahedyehia@yahoo.com.
authors.forename: Nahed
authors.initials: N
authors.lastname: Yehia
authors.affiliation: National Laboratory for Veterinary Quality Control on Poultry Production, Animal Health Research Institute, Dokki, Giza 12618, Egypt.
authors.forename: Abdel-Satar
authors.initials: AS
authors.lastname: Arafa
authors.affiliation: Unit of Infection Models, German Primate Center, Kellnerweg 4, 37077 Goettingen, Germany.
authors.forename: Ahmed
authors.initials: A
authors.lastname: Abd El Wahed
authors.affiliation: Department of Virology, Faculty of Veterinary Medicine, Cairo University, Egypt.
authors.forename: Ahmed A
authors.initials: AA
authors.lastname: El-Sanousi
authors.affiliation: Institutes of Aquaculture, Stirling University, Scotland, UK.
authors.forename: Manfred
authors.initials: M
authors.lastname: Weidmann
authors.affiliation: Department of Virology, Faculty of Veterinary Medicine, Cairo University, Egypt.
authors.forename: Mohamed A
authors.initials: MA
authors.lastname: Shalaby
mesh_terms: D000818:Animals; D004534:Egypt; D019267:Hemagglutinin Glycoproteins, Influenza Virus / Q000032:analysis* / Q000235:genetics; D053124:Influenza A Virus, H5N1 Subtype / Q000235:genetics / Q000302:isolation & purification*; D005585:Influenza in Birds / Q000175:diagnosis*; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D011200:Poultry; D048348:Reverse Transcription; D012680:Sensitivity and Specificity; D013997:Time Factors; D014730:Veterinary Medicine / Q000379:methods*
chemical_list: D019267:Hemagglutinin Glycoproteins, Influenza Virus; C109963:hemagglutinin, avian influenza A virus
keywords: Avian influenza; Real-time RT-PCR; Recombinase polymerase amplification assay; Subtype H5N1