Establishment and application of a PCR-ELISA assay for the detection of seasonal influenza A virus subtypes H1 and H3 and influenza B virus / 中华微生物学和免疫学杂志

ABSTRACT

Objective To develop a PCR-ELISA assay for the rapid, specific and sensitive detec-tion of human seasonal influenza virus ( H1, H3 and B) by using molecular biological and immunological methods in combination.Methods The primers were designed according to the genes encoding the matrix protein ( M) , the H1 and H3 hemagglutinin ( HA) of influenza A virus and the nonstructural proteins ( B-NS) of influenza B virus and then were labeled with biotin.The PCR products were detected by ELISA by use of an internal catching probe labeled with DIG.Results The minimum copy numbers of genes encoding the M, H1, H3 and B-NS proteins detected by the established assay were 1.43?103 , 8.67?102 , 3.86?103 and 5.45?103 copies/μl, respectively, which indicated that the PCR-ELISA assay was about 10 times more sensitive than agarose gel electrophoresis in the detection of PCR products.No cross-reactions between the different subtypes of influenza virus or different species of virus were observed.Moreover, a total of 104 clin-ical specimens of influenza virus were examined by the PCR-ELISA assay, the results of which were consist-ent with those of the virus isolation method.Conclusion The newly developed PCR-ELISA assay was a highly sensitive and specific method for the rapid detection and subtyping of influenza virus, suggesting the possibility of using it in laboratory for the surveillance and detection of influenza virus.