Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
Comparative Study
. 2004 Mar;29(3):179-88.
doi: 10.1016/S1386-6532(03)00122-7.

Design and performance testing of quantitative real time PCR assays for influenza A and B viral load measurement

C L Ward  1 , M H Dempsey, C J A Ring, R E Kempson, L Zhang, D Gor, B W Snowden, M Tisdale
Affiliations
Comparative Study

Design and performance testing of quantitative real time PCR assays for influenza A and B viral load measurement

C L Ward et al. J Clin Virol. 2004 Mar.

Abstract

Background: The antiviral effect of anti-influenza drugs such as zanamivir may be demonstrated in patients as an increased rate of decline in viral load over a time course of treatment as compared with placebo. Historically this was measured using plaque assays, or Culture Enhanced Enzyme Linked Immunosorbent Assay (CE-ELISA).

Objectives: to develop and characterise real time quantitative PCR (qPCR) assays to measure influenza A and B viral load in clinical samples, that offer improvements over existing methods, in particular virus infectivity assays.

Study design: The dynamic range and robustness were established for the real time qPCR assays along with stability of the assay components. Cross validation of the real time PCR assays with CE-ELISA was performed by parallel testing of both serial dilutions of three different subtypes of cultured virus and a panel of influenza positive throat swab specimens.

Results: the assays were specific for influenza A and B and the dynamic ranges were at least seven logs. The assay variability was within acceptable limits but increased towards the lower limit of quantification, which was 3.33 log(10) viral cDNA copies/ml of virus transport medium (ten viral RNA copies/PCR). The components of the assay were robust enough to withstand extended storage and several freeze-thaw cycles. For the real time PCR assays the limit of quantification was equivalent to the virus infectivity cut off, which equates to a 93-fold increase in sensitivity.

Conclusion: Well characterised real time PCR assays offer significant improvements over the existing methods for measuring the viral load of strains of influenza A and B in clinical specimens.

PubMed Disclaimer

Figures

Fig. 1
Fig. 1
Comparison of real time qPCR, CE-ELISA and virus culture over a range of 10-fold serial dilutions of (A) A/Shangdong/3/93 (H3N2), (B) A/Taiwan/1/86 (H1N1) and (C) B/Lisbon/3/96. The virus titre in pfu/ml for the input virus dilutions is estimated from the stock concentration. log values (Y-axis) are given as mean vRNA copies/ml for real time qPCR and TCID50/ml for CE-ELISA. +/− Refers to the presence/absence of haemagglutination activity in virus culture.
Fig. 2
Fig. 2
Comparison of influenza quantification by real time qPCR and CE-ELISA. A subset of 40 patients that were diagnosed influenza A (n=26) or influenza B (n=14) positive and had a complete set of throat swab samples taken on days 1, 3 and 6 were included.
Fig. 3
Fig. 3
Comparison of influenza A vRNA copies/ml in influenza A positive throat swabs taken from subjects with evidence of an upper airway infection, measured by oligonucleotide probe and SYBR Green based real time qPCR assays.
Fig. 4
Fig. 4
Percentage coefficient of variation over the dynamic range of the real time qPCR assays. * Denotes the limit of detection and ** the limit of quantification.
See this image and copyright information in PMC

References

    1. Altschul S.F., Madden T.L., Schäffer A.A., Zhang J., Zhang Z., Miller W., Lipman D.J. Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1999;25:3389–3402. - PMC - PubMed
    1. Barnett J.M., Cadman A., Gor D., Dempsey M., Walters M., Candlin A., Tisdale M., Morley P.J., Owens I.J., Fenton R.J., Lewis A.P., Claas E.C.J., Rimmelzwaan G.F., De Groot R., Osterhaus A.D.M.E. Zanamivir susceptibility monitoring and characterisation of influenza virus clinical isolates obtained during phase II clinical efficacy studies. Antimicrob. Agents Chemother. 2000;44:78–87. - PMC - PubMed
    1. Claas E.C., Osterhaus A.D., van Beek R., De Jong J.C., Rimmelzwaan G.F., Senne D.A., Krauss S., Shortridge K.F., Webster R.G. Human influenza A H5N1 virus related to a highly pathogenic avian influenza virus. Lancet. 1998;351:472–477. - PubMed
    1. De Vries T.J., Fourkour A., Punt C.J.A., van de Locht L.T.F., Wobbes T., van den Bosch S., de Rooij M.J.M., Mensink E.J.B.M., Ruiter D.J., van Muijen G.N.P. Reproducibility of detection of tyrosine and MART-1 transcripts in the peripheral blood of melanoma patients: a quality control study using real-time quantitative RT-PCR. Br. J. Cancer. 1999;80:883–891. - PMC - PubMed
    1. Hayden F.G., Palese P. Influenza virus. In: Rickmann D.D., Whittey R.J., Hayden F.G., editors. Clinical Virology. Churchill Livingstone; New York: 1997.

MeSH terms