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A multiplex real-time RT-PCR for detection and identification of influenza virus types A and B and subtypes H5 and N1

2007; Elsevier BV; Volume: 148; Issue: 1-2 Linguagem: Inglês

10.1016/j.jviromet.2007.10.023

1879-0984

Chunli Wu, Xiao‐Wen Cheng, Jianfan He, Xing Lv, Jingwen Wang, Riqiang Deng, Qingxing Long, Xunzhang Wang,

Viral Infections and Immunology Research

A multiplex real-time RT-PCR method for the simultaneous detection of influenza virus types A and B and identification of subtypes H5 and N1 in a single tube is described. The method was developed with four sets of primers and probes which were specific to influenza virus (sub)types A, B, H5, and N1, and evaluated by using a total of 40 influenza virus reference strains, including 17 avian influenza A (12 H5N1, 1 H1N1, 1 H3N2, 1 H4N6, 1 H7N3, and 1 H9N2), 18 human influenza A (11 H3N2, 6 H1N1 and 1 H5N1) and 5 influenza B viruses. The method exhibited a high specificity and sensitivity of approximately 101–102 copies/μl for each (sub)type and a high reproducibility with intra- and inter-assay CV from 0.13 to 4.24%. In an analysis of 189 clinical samples from patients during the year 2004 and 2005, the method identified 81 positive samples (42.9%) and identified simultaneously 14 type B samples and 11 subtype N1 samples, in comparison only 46 positive samples (24.3%) identified by the conventional culturing method. The method would be a useful molecular diagnostic tool for large-scale screening of clinical samples for influenza virus.