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. 2010 Jan;12(1):74-81.
doi: 10.2353/jmoldx.2010.090095. Epub 2009 Dec 3.

Development of a rapid automated influenza A, influenza B, and respiratory syncytial virus A/B multiplex real-time RT-PCR assay and its use during the 2009 H1N1 swine-origin influenza virus epidemic in Milwaukee, Wisconsin

Eric T Beck  1 , Lisa A Jurgens, Sue C Kehl, Michael E Bose, Teresa Patitucci, Elizabeth LaGue, Patrick Darga, Kimberly Wilkinson, Lorraine M Witt, Jiang Fan, Jie He, Swati Kumar, Kelly J Henrickson
Affiliations

Development of a rapid automated influenza A, influenza B, and respiratory syncytial virus A/B multiplex real-time RT-PCR assay and its use during the 2009 H1N1 swine-origin influenza virus epidemic in Milwaukee, Wisconsin

Eric T Beck et al. J Mol Diagn. 2010 Jan.

Abstract

Rapid, semiautomated, and fully automated multiplex real-time RT-PCR assays were developed and validated for the detection of influenza (Flu) A, Flu B, and respiratory syncytial virus (RSV) from nasopharyngeal specimens. The assays can detect human H1N1, H3N2, and swine-origin (S-OIV) H1N1 Flu A viruses and were effectively used to distinguish Flu A infections (of all subtypes) from Flu B and RSV infections during the current S-OIV outbreak in Milwaukee, WI. The analytical limits of detection were 10(-2) to 10(1) TCID(50)/ml depending on the platform and analyte and showed only one minor cross-reaction among 23 common respiratory pathogens (intermittent cross-reaction to adenovirus at >10(7) TCID(50)/ml). A total of 100 clinical samples were tested by tissue culture, both automated assays, and the US Food and Drug Administration-approved ProFlu+ assay. Both the semiautomated and fully automated assays exhibited greater overall (Flu A, Flu B, and RSV combined) clinical sensitivities (93 and 96%, respectively) and individual Flu A sensitivities (100%) than the Food and Drug Administration-approved test (89% overall sensitivity and 93% Flu A sensitivity). All assays were 99% specific. During the S-OIV outbreak in Milwaukee, WI, the fully automated assay was used to test 1232 samples in 2 weeks. Flu A was detected in 134 clinical samples (126 H1N1 S-OIV, 5 H1N1 [human], and 1 untyped) with 100% positive agreement compared with other "in-house" validated molecular assays, with only 2 false-positive results. Such accurate testing using automated high-throughput molecule systems should allow clinicians and public health officials to react quickly and effectively during viral outbreaks.

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Figures

Figure 1
Figure 1
A: Melt profile of Flu A (green), Flu B (blue), and RSV (red) samples in the FAM channel. B: Melt profile of MS2 in the AP-593 channel.
See this image and copyright information in PMC

References

    1. Fan J, Henrickson KJ, Savatski LL. Rapid simultaneous diagnosis of infections with respiratory syncytial viruses A and B, influenza viruses A and B, and human parainfluenza virus types 1, 2, and 3 by multiplex quantitative reverse transcription-polymerase chain reaction-enzyme hybridization assay (Hexaplex) Clin Infect Dis. 1998;26:1397–1402. - PubMed
    1. Henrickson KJ. Advances in the laboratory diagnosis of viral respiratory disease. Pediatr Infect Dis J. 2004;23(Suppl.):S6–S10. - PubMed
    1. Kehl S, Henrickson KJ. Comparison of three diagnostic methods: hexaplex, tissue culture, and rapid EIA for the detection of respiratory viruses in children. J Clin Microbiol. 2001;39:1696–1701. - PMC - PubMed
    1. Koenig M, Kosha S, Hickman M, Heath D, Riddell S, Aldous S. Detection of influenza virus from throat and pharyngeal swabs with a nested duplex light cycler RT-PCR. Diag Microbiol Inf Dis. 2003;46:35–37. - PubMed
    1. Liolios L, Jenney A, Spelman D, Kotsimbos T, Catton M, Wesselingh S. Comparison of a multiplex reverse transcription-PCR-enzyme hybridization assay with conventional viral culture and immunofluorescence techniques for the detection of seven viral respiratory pathogens. J Clin Microbiol. 2001;39:2779–2783. - PMC - PubMed

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