Molecular detection of a novel human influenza (H1N1) of pandemic potential by conventional and real-time quantitative RT-PCR assays

Abstract

Background: Influenza A viruses are medically important viral pathogens that cause significant mortality and morbidity throughout the world. The recent emergence of a novel human influenza A virus (H1N1) poses a serious health threat. Molecular tests for rapid detection of this virus are urgently needed.

Methods: We developed a conventional 1-step RT-PCR assay and a 1-step quantitative real-time RT-PCR assay to detect the novel H1N1 virus, but not the seasonal H1N1 viruses. We also developed an additional real-time RT-PCR that can discriminate the novel H1N1 from other swine and human H1 subtype viruses.

Results: All of the assays had detection limits for the positive control in the range of 1.0 x 10(-4) to 2.0 x 10(-3) of the median tissue culture infective dose. Assay specificities were high, and for the conventional and real-time assays, all negative control samples were negative, including 7 human seasonal H1N1 viruses, 1 human H2N2 virus, 2 human seasonal H3N2 viruses, 1 human H5N1 virus, 7 avian influenza viruses (HA subtypes 4, 5, 7, 8, 9, and 10), and 48 nasopharyngeal aspirates (NPAs) from patients with noninfluenza respiratory diseases; for the assay that discriminates the novel H1N1 from other swine and human H1 subtype viruses, all negative controls were also negative, including 20 control NPAs, 2 seasonal human H1N1 viruses, 2 seasonal human H3N2 viruses, and 2 human H5N1 viruses.

Conclusions: These assays appear useful for the rapid diagnosis of cases with the novel H1N1 virus, thereby allowing better pandemic preparedness.

Figure 1.

Molecular detection of the novel H1N1 influenza virus by conventional and real-time RT-PCR assays. (A), Amplification of serially diluted positive control samples. The viral RNA inputs in these reactions are indicated in terms of TCID₅₀. M, 1-kb DNA ladder markers (Invitrogen); H₂O, water control; arrow, expected RT-PCR products (173 bp). (B), Amplification of serially diluted positive control samples. The amount of viral RNA (TCID₅₀) used in each of the positive reactions is indicated. (C), Detection of the novel H1N1 from a clinical specimen by the conventional RT-PCR assay. +ve, positive control. (D), Detection of the novel H1N1 from a clinical specimen by the real-time RT-PCR assay. The clinical specimen was serially diluted and tested using the assay. The dilution factors used in each of these positive reactions are indicated. Delta Rn indicates the magnitude of the PCR signal.