Studies on Multiplex RT-PCR for Detection of Avian Influenza Virus Type A Group and Specific H5 and H7 Subtypes

A multiplex Reverse Transcription Polymerase Chain Reaction (mRT-PCR) for Avian Influenza Virus (AIV) has been developed to simultaneously Detect Nucleoprotein (NP) genes common to all AIV subtypes or type A influenza viruses by AIV NP gene or group primers and hemagglutinin (HA) protein genes specific to H5 and H7 subtypes by H5 and H7 primers, respectively. The AIV group primer bases were selected from conserved regions of AIV NP genes determined by sequence comparison and alignment using DNA STAR primer software (DNA STAR Inc., WI). Primers specific to H5 and H7 subtypes were designed on conserved sequences of HA protein genes of H5 and H7 subtypes, respectively. The mRT-PCR products amplified by AIV group primers gave a 750bp fragment, by H7 primers gave a 518bp fragment and by H5 primers gave a 410bp fragment. Evaluation test of the mRTPCR for AIV indicated that all AIV subtypes H1 through H7 and H9 tested were specifically amplified by AIV group primers, H5 subtypes were amplified by both group primers and H5 primers, and H7 subtypes were amplified by both group and H7 primers. Non-H5 or H7 subtypes were not amplified by the use of H5 or H7 primers. Samples contained both H5 and H7 subtypes were amplified by all 3 primer sets of AIV group, H5 and H7 primers simultaneously in one assay. Other avian viruses had no reaction to any of the AIV primers. The mRT-PCR for AIV detected as low as 2 x 10-3 ng of RNA extracted from H7N2 and H5N2 subtypes or as low as 2.2-2.5 Log10 ELD50 of these two viruses. Sensitivity (Se) and Specificity (Sp) of the mRT-PCR for AIV were measured as 92% and 100%, respectively, in comparison with virus isolation in Embryonated Chicken Eggs (ECE).