Simultaneous detection of influenza viruses A and B using real-time quantitative PCR - PubMed (original) (raw)

Comparative Study

Simultaneous detection of influenza viruses A and B using real-time quantitative PCR

L J van Elden et al. J Clin Microbiol. 2001 Jan.

Abstract

Since influenza viruses can cause severe illness, timely diagnosis is important for an adequate intervention. The available rapid detection methods either lack sensitivity or require complex laboratory manipulation. This study describes a rapid, sensitive detection method that can be easily applied to routine diagnosis. This method simultaneously detects influenza viruses A and B in specimens of patients with respiratory infections using a TaqMan-based real-time PCR assay. Primers and probes were selected from highly conserved regions of the matrix protein gene of influenza virus A and the hemagglutinin gene segment of influenza virus B. The applicability of this multiplex PCR was evaluated with 27 influenza virus A and 9 influenza virus B reference strains and isolates. In addition, the specificity of the assay was assessed using eight reference strains of other respiratory viruses (parainfluenza viruses 1 to 3, respiratory syncytial virus Long strain, rhinoviruses 1A and 14, and coronaviruses OC43 and 229E) and 30 combined nose and throat swabs from asymptomatic subjects. Electron microscopy-counted stocks of influenza viruses A and B were used to develop a quantitative PCR format. Thirteen copies of viral RNA were detected for influenza virus A, and 11 copies were detected for influenza virus B, equaling 0.02 and 0.006 50% tissue culture infective doses, respectively. The diagnostic efficacy of the multiplex TaqMan-based PCR was determined by testing 98 clinical samples. This real-time PCR technique was found to be more sensitive than the combination of conventional viral culturing and shell vial culturing.

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Figures

FIG. 1

FIG. 1

Standardization of influenza virus B in the multiplex TaqMan assay. Serial dilutions were made using the EM-counted influenza virus B/Lee/40 stock. A minimum of ±10 copies of RNA could be detected after 40 cycles. The intensity of fluorescence is given on the y axis (ΔRn = reporter signal [FAM]/passive reference signal [ROX]).

FIG. 2

FIG. 2

Standard curve generated by the analysis of known amounts of viral RNA of influenza virus B/Lee/40 with the multiplex TaqMan PCR. Unknown quantities of virus in clinical specimens are plotted against the standard curve.

FIG. 3

FIG. 3

Longitudinal follow-up of six patients with either influenza virus A (patients 3 to 6) or virus B infection (patients 1 and 2). Quantitative analysis was performed using the multiplex TaqMan PCR. The clinical samples were plotted against the standard curve. The filled symbols represent the clinical specimens that were also found positive by conventional culturing and/or shell vial culturing. vp, virus particles.

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