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

Author(s):

Nahed Yehiaa,Abdel-Satar Arafa, Ahmed Abd El Wahed, Ahmed A. El-Sanousi , Manfred Weidmann,Mohamed A. Shalaby

Journal:

Journal of Virological Methods

Year:

2015

Abstract:

The 2006 outbreaks of H5N1 avian influenza in Egypt interrupted poultry production and caused stagger_x005f_x0002_ing 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 ofthe 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.