Development and Evaluation of Real-Time RT-PCR Test for Quantitative and Qualitative Recognition of Current H9N2 Subtype Avian Influenza Viruses in Iran

Avian influenza H9N2 subtype viruses have had a great impact on Iranian industrial poultry production economy since introduction into the country. Rapid and precise identification of the viruses, as well as proper management and epidemiologic studies accompanied with other control measures in poultry industry are of the most important tools for the control and prevention of the disease. To approach this, a real time probe based assay was developed to directly detect a specific influenza virus of H9N2 subtype, which has been endemic in the country over the past two last decades. To prevent laboratory contamination events, it was decided to run a single step rRT-PCR procedure. An Iranian avian influenza virus strain of A/Iran/chicken/772/1998 H9N2 subtype was selected as the reference strain for primers and probe designing. The high agreement value of 99% indicated that the devolved real time assay for the detection of H9 subtype viruses could easily replace the conventional method of virus isolation, particularly in the investigation of viruses like national surveillance plan. The limit of detection was almost one EID50, which was the least real infectious unit that could be detected. Thus, it can be said that this sensitive assay provided provides a powerful tool to not to miss any significant viral biological activity neither in the host body nor in the environment. A high level of correlation coefficient (R2=0.998) also indicated a good correlation between cycle of threshold values and viral concentrations. It can be concluded that the real time RT-PCR could easily replace virus isolation in the detection of H9N2 influenza viruses, especially in large monitoring program. The ability to quantify the virus concentration extends the usage of this test in more accurate studies.