Development and Validation of a One-Step Real-Time PCR Assay for Detection of Subtype H5 Avian Influenza Virus

Objectives: Our purposes from this study was to detect H5 subtype of avian influenza (AI) viruses with high sensitivity and specificity beside rapid test using by real time RT-PCR.Materials & Method: Each components that are effective at reaction includes probe and primer concentration, different materials and term of thermal needs to be optimization. Therefore we performed the multifarious tests to determine optimum concentration for probe and primers and also different thermal cycles, and selected optimum thermal cycle. The specificity of the primer/probe sets was tested on nucleic acids extracted from a diverse array of microorganisms that may be naturally present in samples of avian origin. The sensitivity of the RRT-PCR assay was determined by using in vitro-transcribed RNA and 10-fold serial dilutions of titrated AI viruses.Results & Conclusion: High sensitivity levels were obtained, with limits of detection ranging from 101 to 103 RNA copies and from 101 50% egg infectious dose (EID50)/100 μl to 102.74 EID50/100 μl with titrated viruses. Excellent results were achieved in the intra and inter assay variability tests. The repeatability of the H5 RRT-PCR assay was determined using three different concentrations (high, medium, and low) of viral subtype tested. The coefficients of variation within runs (intra-assay variability) ranged from 0.12% to 2.64%. The inter assay variability was in the range of 2.6% to 4.05%. In conclusion this experiment revealed that real time RT-PCR is more suitable replacement for recognition of highly pathogenic influenza viruses by virus isolation methods.

Keywords:

Subtype , H5 , Real time RT-PCR , Avian Influenza virus