The laboratory detection of a new human influenza A(H1N1)

Objective To establish and optimize a rapid,sensitive and specific assay for new human influenza A(H1N1) detection with the method of RT-PCR.Methods Primers for H1N1 were designed in highly conserved various subtypes and genotypes of influenza virus A and influenza virus B.The sensitivity of the conventional and real-time PCR assays was optimized by evaluating different concentrations of primers and RNA.These assays were simultaneously used to detect H1N1 for influenza-like illness in Huizhou in 2009.Results Specificities of the conventional RT-PCR and real-time PCR assays were high,and seasonal H1N1,H3N2,influenza B virus did not cross-react with influenza A.The detective result of RT-PCR method was 99.5%,which was in accordance with that of real-time PCR method.The conventional RT-PCR assay and real-time PCR assay successfully detected H1N1 clinical specimens,and the RNA from the sample remained detectable in the real-time PCR assay was below 0.5pg.Conclusions This study shows that conventional and real-time quantitative RT-PCR assays are reliable,sensitive and fast for detecting a novel human influenza A(H1N1) subtype and indicate that the constructed method paves the way for the early and rapid detection of H1N1 and quantitative analysis for the infect degree of H1N1.