Evaluation of One step and Two Step RT-PCR approaches using Different types of One-step RT-PCR Commercial kits for Molecular Diagnosis of H9N2

Background/Objectives: H9N2 has been reported from migratory and wild birds in Iran Since 1998, H9N2 outbreaks have been one of the major problems in Iranian poultry industry. Rapid laboratory diagnosis of H9N2 in early stage of disease in farms and in wild birds is very important to improve the prognosis and successfully treatment in industrial farms and have precise surveillance program in migratory and wild birds .The diagnosis of H9N2 is based on the use of molecular tools such as RT-PCR. Methods ::In thus study, We compare and evaluate some one-step RT-PCR kits three One-step RT-PCR kits Qiagen Bioneerand Amersham Bioscience) by WHO designed primers for Iranian H9N2 strains and Two –Steps (RevertAid™ M-MuLV Reverse Transcriptase Taq DNA polymerase Fermentas Co RT-PCR for Molecular Diagnosis of H9N2 Strains After, RNA extraction and standardization cDNA synthesis and PCR done according to the manufacturer’s protocol for the mentioned kits. Results: The best performance observed with more sharp bands and Higher DNA Amplified Product OD) by one step RT-PCR kit in different RNA concentration. Although we observed all of the mentioned kits could detect minimum of RNA template in this experiment. Although we observed all one step RT-PCR kits could detect minimum of RNA template in this experiment. Conclusion :We had more sharp bands by Qiagen one step RT-PCR kit in different RNA concentration. Also These data show that One-Step RT-PCR approach yielded higher rate of H9N2 RNA detection than the Two-Step kit.

Keywords:

Molecular Diagnosis , RT-PCR , H9N2