Sequencing
Avian Orthoavulavirus Serotype-1 F gene cleavage site sequencing
This protocol details the steps to determine and analyse the nucleotide sequence of the cleavage site within the fusion gene of Avian Paramyxovirus Type 1 (APMV-1), including Newcastle Disease Virus (NDV), for diagnosis. Viral RNA is first used to synthesis cDNA which is then amplified by PCR. The PCR amplicons are stained and separated due to size using electrophoresis. The PCR amplicon is excised and purified from the gel using Qiagen gel extraction kit. The amplicon can then be sequenced via the chain termination method using the Big Dye terminator v3.1 cycle sequencing kit and an automated sequencer. The deduced amino acid sequence is used to determine the virulence of the isolate.