Development of reverse transcription recombinase polymerase amplification assay for avian influenza H5N1 HA gene detection

Citation

Yehia N, Arafa A, El Wahed AA, El-Sanousi AA, Weidmann M & Shalaby MA (2015) Development of reverse transcription recombinase polymerase amplification assay for avian influenza H5N1 HA gene detection. Journal of Virological Methods, 223, pp. 45-49. https://doi.org/10.1016/j.jviromet.2015.07.011

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). Anin vitrotranscribed 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 7min, while in real-time RT-PCR, at least 90min 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.

Keywords
Avian influenza; Subtype H5N1; Recombinase polymerase amplification assay; Real-time RT-PCR

Journal
Journal of Virological Methods: Volume 223