Article Citation:
sayed Mohammed abdelwhab, Abdel-satar M. Arafa, Ahmed Erfan, Mona Mehrez Aly, Hafez M. Hafez (2010) Modified H5 real-time RT-PCR oligonucleotides for detection of divergent avian influenza H5N1 viruses in Egypt. Avian Diseases In-Press.
Article
Modified H5 real-time RT-PCR oligonucleotides for detection of divergent avian influenza H5N1 viruses in Egypt
sayed Mohammed abdelwhab1,?, Abdel-satar M. Arafa2, Ahmed Erfan3, Mona Mehrez Aly2, and Hafez M. Hafez4
1 Free University of Berlin
2 National laboratory for veterinary quality control on poultry production
3 National Laboratory for Veterinary Quality Control on Poultry Production, Animal Health Research Institute
4
Abstract
The efforts exerted to prevent circulation of HPAI H5N1 virus in birds are the best way to prevent emergence of new virus subtype with pandemic potential. Despite the blanket vaccination strategy against HPAI H5N1 in Egypt, continuous circulation of the virus in poultry increased since late 2007 due to presence of genetic and antigenic distinct variant strains escape from the immune response of vaccinated birds. Escape of variant strains from detection by real time reverse transcriptase PCR although the suspected poultry flocks have had signs and lesions commonly seen in HPAI H5N1 infected birds were observed. Sequence analysis of these variants revealed multiple single nucleotide substitutions in the primers and probe target the H5 gene by real-time RT-PCR.This study describes the results of RRT-PCR modified from an existing protocol in detection of partial H5 gene segment of the Egyptian H5N1 divergent viruses and applied in the nationwide surveillance. The modified RRT-PCR assay was more sensitive than the original one in detection of Egyptian isolates with 104% amplification efficiency. Sixty-one field samples were found positive in our assay, but only 51 samples tested positive by the original protocol and more sensitive than matrix gene RRT-PCR detection assay. Detection limit of 10 EID50 with the updated oligonucleotides primers and probe set was found. For the foreseeable future, mutation of H5N1 viruses and the endemic situation in developing countries require continuous improvement of current diagnostics to aid containment of the H5N1 virus in poultry sectors and lower the threat of influenza virus spread.
