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2019-9-16 23:20:24

Kim JW, et al. An optimized molecular method for detection of influenza A virus using improved generic primers and concentration of the viral genomic RNA and nucleoprotein complex. J Vet Diagn Invest. 2019 Feb 22
submited by kickingbird at Feb, 24, 2019 19:15 PM from J Vet Diagn Invest. 2019 Feb 22

For reported primer sets used to detect influenza A viruses (IAVs), we verified the nucleotide identities with 9,103 complete sequences of matrix (M) genes. At best, only 93.2% and 85.3% of the sequences had a 100% match with reported forward and reverse primers, respectively. Therefore, we designed new degenerate forward and reverse primers with 100% identity to 94.4% and 96.2% of compared genes, respectively, and the primer set was used with SYBR-based reverse-transcription real-time PCR (SYBR-RT-rtPCR) for lower detection limits. The sensitivity of SYBR-RT-rtPCR with the new primers was 10-fold higher than that with a conventional method in ~2.37% of all M genes in the database used in our study. We successfully increased the sensitivity of SYBR-RT-rtPCR by concentrating the viral ribonucleoprotein (RNP) using immunomagnetic beads and Triton X-100. The improved generic primer set and RNP concentration method may be useful for sensitive detection of IAVs.

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