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International Journal of Virology
Year: 2012  |  Volume: 8  |  Issue: 2  |  Page No.: 203 - 213

Comparative Detection of H5N1 Avian Influenza Virus Using Conventional Rt-PCR and Real Time Rt-PCR

N.M. Hagag, A. Arafa, M.A. Shalaby, A.A. El-Sanousi and M.M. Aly    

Abstract: Highly Pathogenic Avian Influenza (HPAI) caused by influenza A H5N1 virus, poses a significant threat to the poultry industry and humans in Egypt. Since it was first recognized in 2006, the disease has become enzootic in poultry throughout Egypt and still circulates in the poultry population, so the ability to rapidly recognize AIVs in biological specimens is critical for limiting further spread of the disease in poultry. Application of molecular methods such as Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) and Real time RT-PCR (RRT-PCR) as a rapid, specific and sensitive detection methods currently used in national and reference laboratories worldwide. In this study a comparison of the specificity and sensitivity between 2 different formats of conventional RT-PCR (one and two steps) and 3 different formats of RRT-PCR (one step using TaqMan® probe, two steps using TaqMan® probe and two steps using hybridization® probe) were performed and compared as a diagnostic tools for H5N1 virus detection. All these formats of PCRs appeared within the same specificity for H5 gene detection, while they showed difference in sensitivity as the one step conventional RT-PCR showed to be more sensitive than two steps conventional RT-PCR by 10 folds, one step RRT-PCR TaqMan® probe was more sensitive than two steps RRT-PCR TaqMan® probe by 10 folds, two steps RRT-PCR hyberidization® probe is more sensitive than two steps RRT-PCR TaqMan® probe by 100 folds, finally two step RRT-PCR using hyperidization® probe is more sensitive than conventional RT-PCR two steps by 1000 folds. Fifty one field samples were further tested by all mentioned PCR formats the results were the same of that obtained in the sensitivity experiment and agreed with them in placing hybridization probe system of higher sensitivity than TaqMan one step than TaqMan two steps and conventional PCR were of the lowest sensitivity although one step showed higher sensitivity than two steps.

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