Simultaneous one-tube full-length amplification of the NA, NP, M, and NS genes of influenza A viruses for reverse genetics.

Reverse genetics of influenza A viruses has expedited increasingly basic research and vaccine development. Target-primed plasmid amplification using full-length PCR amplicons as inserts was established previously for strain-independent and rapid cloning of all eight influenza A virus genes. This method involves separate amplification of each viral gene using segment-specific primers. Four different primer pairs are required for PCR amplification of the neuraminidase gene depending on the subtype. In order to reduce the number of necessary PCRs, a pair of primers with truncated 3' ends was designed in the present study. This primer pair permitted reliable amplification of the NA, NP, M, and NS genes in one tube whose products can be separated subsequently by their sizes. Full-length amplicons can be generated with this one primer pair from the NA genes of all nine subtypes. By avoiding separate assays for several viral genes, this parallel PCR steps up rapid universal cloning of influenza A virus genes further.