Journal article
Subtyping of Swine Influenza Viruses Using a High-Throughput Real-Time PCR Platform
National Veterinary Institute, Technical University of Denmark1
Virology, Division for Diagnostics & Scientific Advice, National Veterinary Institute, Technical University of Denmark2
Department of Biotechnology and Biomedicine, Technical University of Denmark3
Innate Immunology, Division of Immunology & Vaccinology, National Veterinary Institute, Technical University of Denmark4
Friedrich-Loeffler-Institute5
Influenza A viruses (IAVs) are important human and animal pathogens with high impact on human and animal health. In Denmark, a passive surveillance program for IAV in pigs has been performed since 2011, where screening tests and subsequent subtyping are performed by reverse transcription quantitative real-time PCR (RT-qPCR).
A disadvantage of the current subtyping system is that several assays are needed to cover the wide range of circulating subtypes, which makes the system expensive and time-consuming. Therefore, the aim of the present study was to develop a high-throughput method, which could improve surveillance of swine influenza viruses (swIAVs) and lower the costs of virus subtyping.
Twelve qPCR assays specific for various hemagglutinin and neuraminidase gene lineages relevant for swIAV and six assays specific for the internal genes of IAV were developed and optimized for the high-throughput qPCR platform BioMark (Fluidigm). The qPCR assays were validated and optimized to run under the same reaction conditions using a 48.48 dynamic array (48.48DA).
The sensitivity and specificity was assessed by testing virus isolates and field samples with known subtypes. The results revealed a performance of the swIAV 48.48DA similar to conventional real-time analysis, and furthermore, the specificity of swIAV 48.48DA was very high and without cross reactions between the assays.
This high-throughput system provides a cost-effective alternative for subtyping of swIAVs.
Language: | English |
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Publisher: | Frontiers Media S.A. |
Year: | 2018 |
Pages: | 165 |
ISSN: | 22352988 |
Types: | Journal article |
DOI: | 10.3389/fcimb.2018.00165 |
ORCIDs: | Goecke, Nicole Bakkegård , Hjulsager, Charlotte Kristiane , Skovgaard, Kerstin and Larsen, Lars E. |
Animals DNA Primers Denmark Environmental Monitoring Genome, Viral Hemagglutinins, Viral Humans Influenza A virus Microbiology Neuraminidase Orthomyxoviridae Infections QR1-502 Real-Time Polymerase Chain Reaction Sensitivity and Specificity Swine diagnostics high-throughput real-time PCR real-time PCR subtyping surveillance swine influenza virus