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In silico re-assessment of a diagnostic RT-qPCR assay for universal detection of Influenza A viruses

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  • In silico re-assessment of a diagnostic RT-qPCR assay for universal detection of Influenza A viruses

    Sci Rep. 2019 Feb 7;9(1):1630. doi: 10.1038/s41598-018-37869-w.
    In silico re-assessment of a diagnostic RT-qPCR assay for universal detection of Influenza A viruses.

    Nagy A1,2, Jiřinec T3, Jiřincová H3, Černíková L4, Havlíčková M3.
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    Abstract

    The ongoing evolution of microbial pathogens represents a significant issue in diagnostic PCR/qPCR. Many assays are burdened with false negativity due to mispriming and/or probe-binding failures. Therefore, PCR/qPCR assays used in the laboratory should be periodically re-assessed in silico on public sequences to evaluate the ability to detect actually circulating strains and to infer potentially escaping variants. In the work presented we re-assessed a RT-qPCR assay for the universal detection of influenza A (IA) viruses currently recommended by the European Union Reference Laboratory for Avian Influenza. To this end, the primers and probe sequences were challenged against more than 99,000 M-segment sequences in five data pools. To streamline this process, we developed a simple algorithm called the SequenceTracer designed for alignment stratification, compression, and personal sequence subset selection and also demonstrated its utility. The re-assessment confirmed the high inclusivity of the assay for the detection of avian, swine and human pandemic H1N1 IA viruses. On the other hand, the analysis identified human H3N2 strains with a critical probe-interfering mutation circulating since 2010, albeit with a significantly fluctuating proportion. Minor variations located in the forward and reverse primers identified in the avian and swine data were also considered.


    PMID: 30733500 DOI: 10.1038/s41598-018-37869-w
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