Reverse Transcription Polymerase Chain Reaction
One-step reverse transcription polymerase chain reaction (RT-PCR) was performed in the Avian Influenza Research Laboratory, NVRI, Vom, Plateau State. It was performed using QIAGEN RT-PCR kit targeting the matrix (M) gene in a 9700 thermocycler (Life Technologies, Foster City, CA, USA) as originally described by Spackman et al. (2002). The following modified primers (H5LH1: 5ꞌ ACG TAT GAC TAT CCA CAA TAC TCA G 3ꞌ; H5RH1: 5ꞌ AGA CCA GCT ACC ATG ATT GC 3ꞌ) were used. The following thermal profile was used: a single cycle of reverse transcription for 30 min at 50°C, 2 min at 95°C for reverse transcriptase inactivation and DNA polymerase activation followed by 40 amplification cycles of 15 sec at 95°C and 1 min at 60°C (annealing-extension step). The amplified PCR products were then visualized on 1.5 % agarose gel stained with ethidium bromide.