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A touchdown nucleic acid amplification protocol as an alternative to culture backup for immunofluorescence in the routine diagnosis of acute viral respiratory tract infections

A touchdown nucleic acid amplification protocol as an alternative to culture backup for immunofluorescence in the routine diagnosis of acute viral res

A touchdown nucleic acid amplification protocol as an alternative to culture backup for immunofluorescence in the routine diagnosis of acute viral respiratory tract infections   (Citations: 7)
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BACKGROUND: Immunofluorescence and virus culture are the main methods used to diagnose acute respiratory virus infections. Diagnosing these infections using nucleic acid amplification presents technical challenges, one of which is facilitating the different optimal annealing temperatures needed for each virus. To overcome this problem we developed a diagnostic molecular strip which combined a generic nested touchdown protocol with in-house primer master-mixes that could recognise 12 common respiratory viruses. RESULTS: Over an 18 month period a total of 222 specimens were tested by both immunofluorescence and the molecular strip. The specimens came from 103 males (median age 3.5 y), 80 females (median age 9 y) and 5 quality assurance scheme specimens. Viruses were recovered from a number of specimen types including broncho-alveolar lavage, nasopharyngeal secretions, sputa, post-mortem lung tissue and combined throat and nasal swabs. Viral detection by IF was poor in sputa and respiratory swabs. A total of 99 viruses were detected in the study from 79 patients and 4 quality control specimens: 31 by immunofluorescence and 99 using the molecular strip. The strip consistently out-performed immunofluorescence with no loss of diagnostic specificity. CONCLUSIONS: The touchdown protocol with pre-dispensed primer master-mixes was suitable for replacing virus culture for the diagnosis of respiratory viruses which were negative by immunofluorescence. Results by immunofluorescence were available after an average of 4–12 hours while molecular strip results were available within 24 hours, considerably faster than viral culture. The combined strip and touchdown protocol proved to be a convenient and reliable method of testing for multiple viruses in a routine setting.
Journal: BMC Microbiology - BMC MICROBIOL , vol. 4, no. 1, pp. 41-9, 2004
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    • ...The identity of each isolate was confirmed by a multiplex virus reverse transcriptase (RT)-PCR strip, as previously described [10]...
    • ...Before inclusion, all samples were confirmed negative for a panel of 12 respiratory viruses using a multiplex virus reverse transcriptase (RT)-PCR strip, as previously described [10]...
    • ...Subgroup and genotype analyses of the RSV clinical isolates The standardized multiplex viral RT-PCR strip [10] indicated that all clinical isolates belonged to RSV subgroup A. This was confirmed by G gene sequencing...

    Rémi Villenaveet al. Differential cytopathogenesis of respiratory syncytial virus prototypi...

    • ...It began routine use of a multiplex reverse-transcriptase polymerase chain reaction (RT-PCR) method for virus detection from respiratory samples in July of 2003 [7]...
    • ... of the C. O’Gorman (*) . E. McHenry . P. V. Coyle Regional Virus Laboratory, Royal Group of Hospitals Trust, Belfast BT126BA, Northern Ireland e-mail: ciaran.ogorman@bll.n-i.nhs.uk Tel.: +44-28-90634117 Fax: +44-28-90634803 molecularstripassay.Directcomparisonoftheassaywithan alternative wasnotpossible becauseofthelackof specimens containinghMPVoralternativetests;virusidentificationwas confirmed by selected amplicon sequencing [7]...

    C. O’Gormanet al. Human metapneumovirus in adults: a short case series

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