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

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 log10 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-thawcycles. 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.

A variety of respiratory viruses found in symptomatic travellers returning from countries with ongoing spread of the new influenza A(H1N1)v virus strain

“…Nasopharyngeal swabs were sent to the molecular diagnostic unit at the virological laboratory at Sahlgrenska University Hospital for testing by a multiple real-time PCR targeting 13 viruses and two bacteria, run in six parallel multiplex PCRs on an ABI 7500 instrument [6]. Samples that were reactive for the influenza A component (matrix protein target, [7]) of this PCR were subtyped by an additional real-time PCR targeting the haemagglutinin gene, run in three parallel reactions specific for the H3N2 and H1N1 subtypes that have been circulating for a long time, as well as for the new H1N1v strain (Table 1).…”

West Nile Virus in Golden Eagles, Spain, 2007

“…MCC shares epidemiologic features with Kaposi sarcoma, a malignant tumor associated with human herpesvirus 8 infection (2). In particular, MCC affects predominantly immunocompromised patients such as organ transplant recipients (3,4), patients with B-cell lymphoid tumors (5), and patients with AIDS (6). This similarity between MCC and Kaposi sarcoma may support the hypothesis of an infectious origin of MCC.…”

Diagnostic Methods of Influenza a From Clinical Specimens: A Critical Review of Literature

“…It is emphasized that samples under long storage and constant freezing and thawing will possibly degrade viral RNA and cDNA. This situation may limit the quantification by molecular techniques on samples with low viral copies (Ward et al 2004). …”