Subtyping of swine influenza viruses using a high-throughput real time PCR platform

Nicole Bakkegård Goecke, Jesper Schak Krog, Charlotte Kristiane Hjulsager, Kerstin Skovgaard, T. Harder, Lars Erik Larsen

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    Abstract

    Introduction. Swine influenza is a respiratory disease caused by multiple subtypes of influenza A virus (IAV). The genome of IAV consists of 8 segments and subtype classification is based on the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). In Denmark, the influenza screening test and subsequent subtyping is performed by real time RT-PCR (RT-qPCR) but several assays are needed to cover the wide range of circulating subtypes which is expensive,resource and time demanding. To mitigate these restrictions the high-throughput qPCR platform BioMark (Fluidigm) has been explored. The BioMark platform uses less sample and reagent volume compared to standard qPCR platforms and allows for up to 9,216 parallel reactions on one chip. Materials and methods. A total of 14 PCR assays specific for the different subtypes of HA and NA genes relevant for swine influenza and 6 assays specific for the internal genes of IAV were validated and optimised to run under identical reaction conditions and assembled on a dynamic array chip (Fluidigm). Results. The sensitivity and specificity of the chip was assessed by testing cell culture isolates and field samples with known subtypes (based on sequencing). The results revealed that the performance of the dynamic chip was similar to conventional real time analysis. Discussion and conclusion. Application of the chip for subtyping of swine influenza has resulted in a significant reduction in time, cost and working hours. Thereby, it is possible to offer diagnostic services with reduced price and turnover time which will facilitate choice of vaccines and by that lead to reduction of antibiotic used.
    Original languageEnglish
    Publication date2017
    Publication statusPublished - 2017
    Event9th European Symposium of Porcine Health Management (ESPHM 2017) - Prague, Czech Republic
    Duration: 3 May 20175 May 2017
    Conference number: 9
    http://www.esphm2017.org/

    Conference

    Conference9th European Symposium of Porcine Health Management (ESPHM 2017)
    Number9
    CountryCzech Republic
    CityPrague
    Period03/05/201705/05/2017
    Internet address

    Cite this

    Goecke, N. B., Krog, J. S., Hjulsager, C. K., Skovgaard, K., Harder, T., & Larsen, L. E. (2017). Subtyping of swine influenza viruses using a high-throughput real time PCR platform. Abstract from 9th European Symposium of Porcine Health Management (ESPHM 2017), Prague, Czech Republic.
    Goecke, Nicole Bakkegård ; Krog, Jesper Schak ; Hjulsager, Charlotte Kristiane ; Skovgaard, Kerstin ; Harder, T. ; Larsen, Lars Erik. / Subtyping of swine influenza viruses using a high-throughput real time PCR platform. Abstract from 9th European Symposium of Porcine Health Management (ESPHM 2017), Prague, Czech Republic.
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    title = "Subtyping of swine influenza viruses using a high-throughput real time PCR platform",
    abstract = "Introduction. Swine influenza is a respiratory disease caused by multiple subtypes of influenza A virus (IAV). The genome of IAV consists of 8 segments and subtype classification is based on the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). In Denmark, the influenza screening test and subsequent subtyping is performed by real time RT-PCR (RT-qPCR) but several assays are needed to cover the wide range of circulating subtypes which is expensive,resource and time demanding. To mitigate these restrictions the high-throughput qPCR platform BioMark (Fluidigm) has been explored. The BioMark platform uses less sample and reagent volume compared to standard qPCR platforms and allows for up to 9,216 parallel reactions on one chip. Materials and methods. A total of 14 PCR assays specific for the different subtypes of HA and NA genes relevant for swine influenza and 6 assays specific for the internal genes of IAV were validated and optimised to run under identical reaction conditions and assembled on a dynamic array chip (Fluidigm). Results. The sensitivity and specificity of the chip was assessed by testing cell culture isolates and field samples with known subtypes (based on sequencing). The results revealed that the performance of the dynamic chip was similar to conventional real time analysis. Discussion and conclusion. Application of the chip for subtyping of swine influenza has resulted in a significant reduction in time, cost and working hours. Thereby, it is possible to offer diagnostic services with reduced price and turnover time which will facilitate choice of vaccines and by that lead to reduction of antibiotic used.",
    author = "Goecke, {Nicole Bakkeg{\aa}rd} and Krog, {Jesper Schak} and Hjulsager, {Charlotte Kristiane} and Kerstin Skovgaard and T. Harder and Larsen, {Lars Erik}",
    year = "2017",
    language = "English",
    note = "9th European Symposium of Porcine Health Management (ESPHM 2017), ESPHM ; Conference date: 03-05-2017 Through 05-05-2017",
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    Goecke, NB, Krog, JS, Hjulsager, CK, Skovgaard, K, Harder, T & Larsen, LE 2017, 'Subtyping of swine influenza viruses using a high-throughput real time PCR platform' 9th European Symposium of Porcine Health Management (ESPHM 2017), Prague, Czech Republic, 03/05/2017 - 05/05/2017, .

    Subtyping of swine influenza viruses using a high-throughput real time PCR platform. / Goecke, Nicole Bakkegård; Krog, Jesper Schak; Hjulsager, Charlotte Kristiane; Skovgaard, Kerstin; Harder, T.; Larsen, Lars Erik.

    2017. Abstract from 9th European Symposium of Porcine Health Management (ESPHM 2017), Prague, Czech Republic.

    Research output: Contribution to conferenceConference abstract for conferenceResearchpeer-review

    TY - ABST

    T1 - Subtyping of swine influenza viruses using a high-throughput real time PCR platform

    AU - Goecke, Nicole Bakkegård

    AU - Krog, Jesper Schak

    AU - Hjulsager, Charlotte Kristiane

    AU - Skovgaard, Kerstin

    AU - Harder, T.

    AU - Larsen, Lars Erik

    PY - 2017

    Y1 - 2017

    N2 - Introduction. Swine influenza is a respiratory disease caused by multiple subtypes of influenza A virus (IAV). The genome of IAV consists of 8 segments and subtype classification is based on the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). In Denmark, the influenza screening test and subsequent subtyping is performed by real time RT-PCR (RT-qPCR) but several assays are needed to cover the wide range of circulating subtypes which is expensive,resource and time demanding. To mitigate these restrictions the high-throughput qPCR platform BioMark (Fluidigm) has been explored. The BioMark platform uses less sample and reagent volume compared to standard qPCR platforms and allows for up to 9,216 parallel reactions on one chip. Materials and methods. A total of 14 PCR assays specific for the different subtypes of HA and NA genes relevant for swine influenza and 6 assays specific for the internal genes of IAV were validated and optimised to run under identical reaction conditions and assembled on a dynamic array chip (Fluidigm). Results. The sensitivity and specificity of the chip was assessed by testing cell culture isolates and field samples with known subtypes (based on sequencing). The results revealed that the performance of the dynamic chip was similar to conventional real time analysis. Discussion and conclusion. Application of the chip for subtyping of swine influenza has resulted in a significant reduction in time, cost and working hours. Thereby, it is possible to offer diagnostic services with reduced price and turnover time which will facilitate choice of vaccines and by that lead to reduction of antibiotic used.

    AB - Introduction. Swine influenza is a respiratory disease caused by multiple subtypes of influenza A virus (IAV). The genome of IAV consists of 8 segments and subtype classification is based on the surface glycoproteins hemagglutinin (HA) and neuraminidase (NA). In Denmark, the influenza screening test and subsequent subtyping is performed by real time RT-PCR (RT-qPCR) but several assays are needed to cover the wide range of circulating subtypes which is expensive,resource and time demanding. To mitigate these restrictions the high-throughput qPCR platform BioMark (Fluidigm) has been explored. The BioMark platform uses less sample and reagent volume compared to standard qPCR platforms and allows for up to 9,216 parallel reactions on one chip. Materials and methods. A total of 14 PCR assays specific for the different subtypes of HA and NA genes relevant for swine influenza and 6 assays specific for the internal genes of IAV were validated and optimised to run under identical reaction conditions and assembled on a dynamic array chip (Fluidigm). Results. The sensitivity and specificity of the chip was assessed by testing cell culture isolates and field samples with known subtypes (based on sequencing). The results revealed that the performance of the dynamic chip was similar to conventional real time analysis. Discussion and conclusion. Application of the chip for subtyping of swine influenza has resulted in a significant reduction in time, cost and working hours. Thereby, it is possible to offer diagnostic services with reduced price and turnover time which will facilitate choice of vaccines and by that lead to reduction of antibiotic used.

    M3 - Conference abstract for conference

    ER -

    Goecke NB, Krog JS, Hjulsager CK, Skovgaard K, Harder T, Larsen LE. Subtyping of swine influenza viruses using a high-throughput real time PCR platform. 2017. Abstract from 9th European Symposium of Porcine Health Management (ESPHM 2017), Prague, Czech Republic.