Multiplex nested RT-PCR for detecting avian influenza virus, infectious bronchitis virus and Newcastle disease virus

► This is the first study to improve the detection limit of multiplex PCR in poultry. ► The increased sensitivities of this technique were 104.9, 101.9 and 104 EID50/ml for AIV, IBV and NDV, respectively. ► This method combines the simultaneous detection of multiplex PCR with the higher sensitivity of nested PCR.