Influenza virus types and subtypes detection by single step single tube multiplex reverse transcription-polymerase chain reaction (RT-PCR) and agarose gel electrophoresis

Abstract

Influenza virus type and subtype specific primers were selected for use in reverse transcription polymerase chain reaction (RT-PCR). The selected primer sets were used in a single step RT-PCR of influenza virus RNA in multiplex format for the detection of virus type and subtypes. Three one step reaction conditions are optimized: (1) multiplex typing only, (2) multiplex subtyping of influenza A, and (3) multiplex typing and subtyping simultaneously. RNA from strains of influenza virus type A of subtypes H1N1, H3N2 and H5N1 and influenza virus type B was used for amplification and detection. The amplified DNA fragments were size analyzed for the presence of specific type and subtypes target DNA by agarose gel electrophoresis. The sensitivity of detection was about 0.01 TCID(50) for both influenza type A and B when amplification was for typing alone. In multiplex subtyping or multiplex typing and subtyping simultaneously, the sensitivities for types and subtypes specific bands were 0.01-0.1 TCID (50) in the tested volume.