Development of duplex and triplex conventional RT-PCR for the detection of H5N1 avian influenza virus

Molecular diagnostic tests are commonly used to diagnose avian influenza virus because they are sensitive, rapid and cost effective. However, continuous shift and drift of avian influenza viruses and H5N1 in particular, is a real challenge for development of specific genetic based detection techniques. Furthermore, most of current molecular diagnostic commercial kits rely on the detection of H5 and/or N1 genes without the presence of the M1 gene which represents the most conservative gene in the type A avian influenza viruses (AIV). This enable spread of undetected mutants. In the present investigation we have optimized a primer set for polymerase chain reaction (PCR)-based detection of influenza A viruses H5, N1 and M1 genes that was validated with a panel of influenza A virus reference strains representing H5, H7, H9 and H13. Specificity test was carried out by the use of eight type A AN subtypes (H5N1, H5N2, H5N9, H7N1, H7N3, H7N7, H9N2 and H13N6). Results showed that this protocol is capable of produce two distinguished bands represents the H5 and N1 genes in the duplex format. While, it generated three distinguished bands represents the H5, N1 and M1 genes (in the triplex format). Moreover, the specificity test showed that the used primers do not have cross-reactivity with the AN subtypes other than H5N1. In addition, Sensitivity test revealed that the duplex format has a similar sensitivity limit as the triplex one.