Amplification of the entire genome of influenza A virus H1N1 and H3N2 subtypes by reverse-transcription polymerase chain reaction.
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 3'-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 H1N1 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.
Chan
Chi-Ho
CH
Department of Microbiology and Immunology, Chung Shan Medical University (CSMU), 110, Sec. No. 1, Chien-Kuo N. Road, Taichung 402, Taiwan, Republic of China. chiho@csmu.edu.tw
Lin
Keh-Liang
KL
Chan
You
Y
Wang
Ya-Li
YL
Chi
Yu-Tsai
YT
Tu
Hsiao-Li
HL
Shieh
Happy-K
HK
Liu
Wu-Tse
WT