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

• Published in: Journal of clinical virology Vol. 29; no. 3; pp. 179 - 188
• Main Authors: Ward, C.L, Dempsey, M.H, Ring, C.J.A, Kempson, R.E, Zhang, L, Gor, D, Snowden, B.W, Tisdale, M
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 01.03.2004; Elsevier Science
• Subjects: Biological and medical sciences; DNA, Complementary - genetics; Enzyme-Linked Immunosorbent Assay; Fundamental and applied biological sciences. Psychology; Human viral diseases; Humans; Infectious diseases; Influenza A virus - genetics; Influenza A virus - isolation & purification; Influenza B virus - genetics; Influenza B virus - isolation & purification; Influenza virus; Influenza, Human - virology; Medical sciences; Microbiology; Miscellaneous; Pharynx - virology; Polymerase Chain Reaction - instrumentation; Polymerase Chain Reaction - methods; Real-time PCR; Reproducibility of Results; RNA, Viral - analysis; Sensitivity and Specificity; Specimen Handling; Validation; Viral diseases; Viral Load; Virology; Virus Cultivation; Validation; Real-time PCR; Influenza virus; Microbiology; Real time; Virology; Infection; Polymerase chain reaction; Viral disease; Influenza A; Viral load; Influenza B; Quantitative analysis
• ISSN: 1386-6532; 1873-5967
• DOI: 10.1016/S1386-6532(03)00122-7

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.