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      Multiplex real-time PCR for detection of respiratory tract infections

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          Abstract

          Background

          Broad diagnostics of respiratory infection by molecular assays has not yet won acceptance due to technical difficulties and high costs.

          Objectives

          To evaluate clinical applicability of multiplex real-time PCR.

          Study design

          An assay targeting influenza virus A (IfA) and B (IfB), parainfluenza 1-3 (PIV), human metapneumovirus (MPV), respiratory syncytial virus (RSV), rhinovirus (RV), enterovirus (EV), adenovirus (AdV), human coronaviruses (229E, OC43, NL63), M. pneumoniae and Ch. pneumoniae was developed and run daily on consecutive clinical nasopharyngeal swab samples.

          Results

          An etiology was identified in 48% of the 954 samples, with IfA in 25%, RV in 20%, MPV in 10% and M. pneumoniae in 10% of the positive. By a rational procedure costs could be reduced and the customer price set relatively low (€33 per sample).

          Conclusion

          Streamlined testing and cost limitation is achievable and probably critical for implementation of a broad molecular diagnostics of respiratory infections.

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          Most cited references14

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          Rapid and quantitative detection of human adenovirus DNA by real-time PCR.

          Rapid diagnosis of human adenovirus (HAdV) infections was achieved by PCR in the recent years. However, conventional PCR has the risk of carry-over contamination due to open handling with its products, and results are only qualitative. Therefore, a quantitative "real-time" PCR with consensus primer and probe (dual fluorescence labelled, "TaqMan") sequences for a conserved region of the hexon gene was designed and evaluated. Real-time PCR detected all 51 HAdV prototypes. Sensitivity of the assay was
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            Rapid and sensitive method using multiplex real-time PCR for diagnosis of infections by influenza a and influenza B viruses, respiratory syncytial virus, and parainfluenza viruses 1, 2, 3, and 4.

            Laboratory diagnosis of viral respiratory infections is generally performed by virus isolation in cell culture and immunofluorescent assays. Reverse transcriptase PCR is now recognized as a sensitive and specific alternative for detection of respiratory RNA viruses. A rapid real-time multiplex PCR assay was developed for the detection of influenza A and influenza B viruses, human respiratory syncytial virus (RSV), parainfluenza virus 1 (PIV1), PIV2, PIV3, and PIV4 in a two-tube multiplex reaction which used molecular beacons to discriminate the pathogens. A total of 358 respiratory samples taken over a 1-year period were analyzed by the multiplex assay. The incidence of respiratory viruses detected in these samples was 67 of 358 (19%) and 87 of 358 (24%) by culture and real-time PCR, respectively. Culture detected 3 influenza A virus, 2 influenza B virus, 57 RSV, 2 PIV1, and 2 PIV3 infections. All of these culture-positive samples and an additional 5 influenza A virus, 6 RSV, 2 PIV1, 1 PIV2, 1 PIV3, and 3 PIV4 infections were detected by the multiplex real-time PCR. The application of real-time PCR to clinical samples increases the sensitivity for respiratory viral diagnosis. In addition, results can be obtained within 6 h, which increases clinical relevance. Therefore, use of this real-time PCR assay would improve patient management and infection control.
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              Development of three multiplex RT-PCR assays for the detection of 12 respiratory RNA viruses

              Three multiplex hemi-nested RT-PCR assays were developed to detect simultaneously 12 RNA respiratory viruses: influenza viruses A, B and C, human respiratory syncytial virus (hRSV), human metapneumovirus (hMPV), parainfluenza virus types 1–4 (PIV-1, -2, -3 and -4), human coronavirus OC43 and 229E (HCoV) and rhinovirus (hRV). An internal amplification control was included in one of the RT-PCR assays. The RT-PCR multiplex 1 and the hemi-nested multiplex 1 detected 1 and 0.1 TCID50 of RSV A, respectively, and 0.01 and 0.001 TCID50 of influenza virus A/H3N2, respectively. Two hundred and three nasal aspirates from hospitalised children were retrospectively tested in comparison with two conventional methods: direct immunofluorescence assay and viral isolation technique. Almost all samples (89/91) that were positive by immunofluorescence assay and/or viral isolation technique were detected by the multiplex assay. This method also detected an additional 85 viruses and 33 co-infections. The overall sensitivity (98%), rapidity and enhanced efficiency of these multiplex hemi-nested RT-PCR assays suggest that they would be a significant improvement over conventional methods for the detection of a broad spectrum of respiratory viruses.
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                Author and article information

                Contributors
                Journal
                J Clin Virol
                J. Clin. Virol
                Journal of Clinical Virology
                Elsevier B.V.
                1386-6532
                1873-5967
                26 December 2007
                January 2008
                26 December 2007
                : 41
                : 1
                : 53-56
                Affiliations
                Department of Infection, the Sahlgrenska Academy, Göteborg University, 416 85 Göteborg, Sweden
                Author notes
                [* ]Corresponding author. Tel.: +46 73 8000 400. Robin.bl@ 123456telia.com
                Article
                S1386-6532(07)00394-0
                10.1016/j.jcv.2007.10.029
                7172039
                18093871
                50275d54-1a9d-4c1e-8d3a-f3f3b2d0f831
                Copyright © 2007 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.

                Categories
                Article

                Microbiology & Virology
                molecular diagnostics,respiratory infection,real-time pcr,taqman
                Microbiology & Virology
                molecular diagnostics, respiratory infection, real-time pcr, taqman

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