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Abstract:
Background: Negative sence RNA genome of Influenza A virus contains 8 segments coding for 12-14 proteins depending on strains. Genetically modified virus is caused world wide spread of a new Influenza in the human population. Developing a rapid and accurate diagnostic method to identify new species is necessary. The aim of this study was rapid detection of new species of Influenza A subtypes using specific RT-PCR based on hemagglutinin gene.
Methods: In this study 30 Nasopharynx samples of patient cultured in embryonated eggs. Then RNA was extracted, cDNA prepared and PCR was performed using specific primers designed from hemagglutinin gene. PCR products purified and sequenced.
Findings: PCR products sequences compared with Influenza A sequences obtained from the Gene Bank database. All positive isolates most closely related to the influenza reference strain. This Result showed that the specific RT-PCR used was able to amplified and detect Influenza A subtypes from clinical specimen.
Conclusion: The results of this study confirmed that PCR based on hemagglutinin gene with sequencing is a sensitive and accurate method for rapid detection of influenza A new subtypes directly from clinical specimen which is useful in preparation and production of vaccine.

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Objective: Influenza virus A (H1N1) is an important subtype of the influenza respiratory viruses, which has important worldwide implications. Hemagglutinin (HA), an important viral antigen, is responsible for binding to human cell receptors leading to an onset of the disease process. Considering the critical role of viral attachment, this study focuses on the extraction and cloning of HA and its large subunit HA1 genes to generate recombinant baculovirus shuttle vectors (bacmid) in order to produce recombinant proteins in insect cells. Methods: Human influenza virus A/New Caledonia 99/20/(H1N1) was propagated in MDCK cell culture. Total viral RNA was extracted using easy-red solution. The full-length HA genome and HA1 fragment were amplified by RT- PCR using specific primers, cloned into a pGEM®-TEasy vector, and then subcloned into a pFastBac HT plasmid. Finally, recombinant bacmids that contained the genes of interest were produced in E. coli DH10Bac™ cells. Results: Expected PCR products of HA genes were evaluated through gel electrophoresis and restriction enzyme analysis. Recombinant pGEM®-TEasy vectors and pFastBac HT donor plasmids were confirmed by PCR, digestion, and sequencing. Construction of recombinant bacmid DNA was verified by using blue-white colony screening, overnight electrophoresis, and PCR analysis that used either pUC/M13 or gene-specific primers. Conclusion: In this study, we have successfully constructed recombinant Bacmid DNA that encoded the full-length HA genome and its HA1 subunit. We intend to transfect sf9 insect cells with these constructs to generate recombinant baculovirus and produce large amounts of desired proteins for future studies.