An optimized molecular method for detection of influenza A virus using improved generic primers and concentration of the viral genomic RNA and nucleoprotein complex

Ji Woon Kim, Chung Young Lee, Thanh Trung Nguyen, Il Hwan Kim, Hyuk Joon Kwon, Jae Hong Kim

Research output: Contribution to journalArticlepeer-review

Abstract

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.

Original languageEnglish
Pages (from-to)175-183
Number of pages9
JournalJournal of Veterinary Diagnostic Investigation
Volume31
Issue number2
DOIs
StatePublished - 1 Mar 2019

Keywords

  • Generic primer
  • influenza A virus
  • matrix gene
  • real-time PCR
  • ribonucleoprotein

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