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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. Lin, K.L. Chan, Y. Wang, Y.L. Chi, Y.T. Tu, H.L. Shieh, H.K. Liu, W.T. |
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. |
URI: | http://hdl.handle.net/11455/70550 | ISSN: | 0166-0934 | DOI: | 10.1016/j.jviromet.2006.03.027 |
Appears in Collections: | 期刊論文 |
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