Multiplex RT-PCR for the rapid detection of matrix protein and H5 genes of highly pathogenic avian influenza virus

Avian influenza (AI) is an economic, zoonotically important disease in worldwide. It is an infectious disease of birds caused by influenza virus which also affect human beings. Outbreaks of H 5 N 1 highly pathogenic avian influenza (HPAI) virus caused great economic losses to the poultry industry. Rapid typing and subtyping of HPAI viruses are desirable for taking prompt control measure to prevent the spread of the disease. Here, we develop multiplex RT-PCR technique for the rapid, simultaneous detection of Matrix protein (MP) and H 5 genes of HPAI from RNA extracted from tracheal swabs using Finnzymes Phusion TM Flash High-Fidelity PCR Master Mix (2x) based on modified Phusion Hot Start DNA Polymerase and QIAgen RT-PCR Kit. Reverse transcription and PCR were carried out with a mixture of primers specific for influenza viruses of type A subtype H 5 in a single reaction system under identical conditions. Both the PCR detection systems amplified matrix protein and H 5 genes specific 245bp and 545bp bands, respectively. However, QIAgen RT-PCR Kit required 3 hours to complete the PCR cycle, whereas Phusion Flash method required 1 hour and 15 minutes only. On the other hand, multiplex PCR was finished in one tube for both MP and H 5 genes. Therefore, it is time and cost effective. Phusion Flash method required relatively less time and cost. After validation with few more field samples, it can be used for the rapid, simultaneous detection of MP and H 5 genes of HPAI in the field condition.