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|標題:||Amplification of the entire genome of influenza A virus H1N1 and H3N2 subtypes by reverse-transcription polymerase chain reaction||作者:||Chan, C.H.
|關鍵字:||influenza A virus;entire genome;Thermoscript (TM) reverse;transcriptase;RT-PCR;molecular epidemiology;nucleoprotein gene;rt-pcr;evolution;differentiation;sequences;origin;primer;china;set||Project:||Journal of Virological Methods||期刊/報告no：:||Journal of Virological Methods, Volume 136, Issue 1-2, Page(s) 38-43.||摘要:||
This study describes the development of a simple RT-PCR method to amplify the whole genome of the influenza A virus based on the amplification of full-length gene segments. Primers were designed based on the conserved regions of both the 5'-end and the Y-end of each gene segment. After optimizing the duration and temperature of denaturing, annealing, and extension, these primers could amplify all of the full-length gene segments. To test the accuracy of the method, all amplicons were subjected to DNA sequencing with an autosequencer. Eighteen strains of influenza A virus which belonged to HIM or H3N2 subtypes were tested. All eight segments of both subtypes were successfully amplified in all tested strains. Using a newly developed reverse-transcriptase (RT), primers and PCR running conditions, this study established a protocol to amplify the entire genome of the influenza A virus. This method provides a tool which can be used for the amplification of all genes of the H1N1 and H3N2 subtypes of influenza A virus prior to analysis of their sequences, and to construct expression plasmids for further study. (c) 2006 Elsevier B.V. All rights reserved.
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