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

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 Imm...

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Autores principales: Ward, C.L, Dempsey, M.H, Ring, C.J.A, Kempson, R.E, Zhang, L, Gor, D, Snowden, B.W, Tisdale, M
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier B.V. 2004
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7128145/
https://www.ncbi.nlm.nih.gov/pubmed/14962787
http://dx.doi.org/10.1016/S1386-6532(03)00122-7
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author Ward, C.L
Dempsey, M.H
Ring, C.J.A
Kempson, R.E
Zhang, L
Gor, D
Snowden, B.W
Tisdale, M
author_facet Ward, C.L
Dempsey, M.H
Ring, C.J.A
Kempson, R.E
Zhang, L
Gor, D
Snowden, B.W
Tisdale, M
author_sort Ward, C.L
collection PubMed
description 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.
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spelling pubmed-71281452020-04-08 Design and performance testing of quantitative real time PCR assays for influenza A and B viral load measurement Ward, C.L Dempsey, M.H Ring, C.J.A Kempson, R.E Zhang, L Gor, D Snowden, B.W Tisdale, M J Clin Virol Article 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. Elsevier B.V. 2004-03 2003-07-15 /pmc/articles/PMC7128145/ /pubmed/14962787 http://dx.doi.org/10.1016/S1386-6532(03)00122-7 Text en Copyright © 2003 Elsevier B.V. All rights reserved. Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.
spellingShingle Article
Ward, C.L
Dempsey, M.H
Ring, C.J.A
Kempson, R.E
Zhang, L
Gor, D
Snowden, B.W
Tisdale, M
Design and performance testing of quantitative real time PCR assays for influenza A and B viral load measurement
title Design and performance testing of quantitative real time PCR assays for influenza A and B viral load measurement
title_full Design and performance testing of quantitative real time PCR assays for influenza A and B viral load measurement
title_fullStr Design and performance testing of quantitative real time PCR assays for influenza A and B viral load measurement
title_full_unstemmed Design and performance testing of quantitative real time PCR assays for influenza A and B viral load measurement
title_short Design and performance testing of quantitative real time PCR assays for influenza A and B viral load measurement
title_sort design and performance testing of quantitative real time pcr assays for influenza a and b viral load measurement
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7128145/
https://www.ncbi.nlm.nih.gov/pubmed/14962787
http://dx.doi.org/10.1016/S1386-6532(03)00122-7
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