Multiplex Assay for Simultaneously Typing and Subtyping Influenza Viruses by Use of an Electronic Microarray

Abstract

We report on the use of an electronic microarray to simultaneously type influenza A and B viruses and to distinguish influenza A virus subtypes H1N1 and H3N2 from the potentially pandemic avian virus subtype H5N1. The assay targets seven genes: the H1, H3, H5, N1, and N2 genes of influenza A virus; the matrix protein M1 gene of influenza A virus; and the nonstructural protein (NS) gene of influenza B virus. By combining a two-step reverse transcription-multiplex PCR with typing and subtyping on the electronic microarray, the assay achieved an analytical sensitivity of 10 2 to 10 3 copies of transcripts per reaction for each of the genes. The assay correctly typed and subtyped 15 different influenza virus isolates, including two influenza B virus, five A/H1N1, six A/H3N2, and two A/H5N1 isolates. In addition, the assay correctly identified 8 out of 10 diluted, archived avian influenza virus specimens with complete typing and subtyping information and 2 specimens with partial subtyping information. In a study of 146 human clinical specimens that had previously been shown to be positive for influenza virus or another respiratory virus, the assay showed a clinical sensitivity of 96% and a clinical specificity of 100%. The assay is a rapid, accurate, user-friendly method for simultaneously typing and subtyping influenza viruses.

Rapid Multiplex Reverse Transcription-PCR Typing of Influenza A and B Virus, and Subtyping of Influenza A Virus into H1, 2, 3, 5, 7, 9, N1 (Human), N1 (Animal), N2, and N7, Including Typing of Novel Swine Origin Influenza A (H1N1) Virus, during the 2009 Outbreak in Milwaukee, Wisconsin

“…Three microarray-based assays have been developed for the subtyping of influenza viruses. The first was developed by our laboratory (13) and was a precursor to our present assay. This assay uses a multiplex RT-PCR procedure for amplification, followed by detection with an automated electronic microarray platform (NC-400; Nanogen Inc., San Diego, CA).…”

Rapid Semiautomated Subtyping of Influenza Virus Species during the 2009 Swine Origin Influenza A H1N1 Virus Epidemic in Milwaukee, Wisconsin

“…A goal of our laboratory has been to develop rapid, sensitive, and specific semiautomated and automated multiplex assays for the detection of common community-acquired respiratory viruses (5,9,11,14). Tissue culture had been the "gold standard" for respiratory virus detection until approximately the late 1990s, when large multiplex RT-PCR assays first became available clinically and commercially (5).…”

Impact of Microarray Technology in Influenza Virus Research and Diagnostics

“…Overall, 20 to 50-mer probes are used to identify each subtype of HA and NA. With more development more advanced electronic microchips with more specific shorter probes and automated detection system were used for influenza virus sub typing [22,23].…”