tetano
Editor, Senior Moderator
J Vet Diagn Invest. 2019 Feb 22:1040638719830760. doi: 10.1177/1040638719830760. [Epub ahead of print]
[h=1]An optimized molecular method for detection of influenza A virus using improved generic primers and concentration of the viral genomic RNA and nucleoprotein complex.[/h] Kim JW[SUP]1,[/SUP][SUP]2,[/SUP][SUP]3,[/SUP][SUP]4,[/SUP][SUP]5[/SUP], Lee CY[SUP]1,[/SUP][SUP]2,[/SUP][SUP]3,[/SUP][SUP]4,[/SUP][SUP]5[/SUP], Nguyen TT[SUP]1,[/SUP][SUP]2,[/SUP][SUP]3,[/SUP][SUP]4,[/SUP][SUP]5[/SUP], Kim IH[SUP]1,[/SUP][SUP]2,[/SUP][SUP]3,[/SUP][SUP]4,[/SUP][SUP]5[/SUP], Kwon HJ[SUP]1,[/SUP][SUP]2,[/SUP][SUP]3,[/SUP][SUP]4,[/SUP][SUP]5[/SUP], Kim JH[SUP]1,[/SUP][SUP]2,[/SUP][SUP]3,[/SUP][SUP]4,[/SUP][SUP]5[/SUP].
[h=3]Author information[/h]
[h=3]Abstract[/h] 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.
[h=4]KEYWORDS:[/h] Generic primer; influenza A virus; matrix gene; real-time PCR; ribonucleoprotein
PMID: 30795722 DOI: 10.1177/1040638719830760
[h=1]An optimized molecular method for detection of influenza A virus using improved generic primers and concentration of the viral genomic RNA and nucleoprotein complex.[/h] Kim JW[SUP]1,[/SUP][SUP]2,[/SUP][SUP]3,[/SUP][SUP]4,[/SUP][SUP]5[/SUP], Lee CY[SUP]1,[/SUP][SUP]2,[/SUP][SUP]3,[/SUP][SUP]4,[/SUP][SUP]5[/SUP], Nguyen TT[SUP]1,[/SUP][SUP]2,[/SUP][SUP]3,[/SUP][SUP]4,[/SUP][SUP]5[/SUP], Kim IH[SUP]1,[/SUP][SUP]2,[/SUP][SUP]3,[/SUP][SUP]4,[/SUP][SUP]5[/SUP], Kwon HJ[SUP]1,[/SUP][SUP]2,[/SUP][SUP]3,[/SUP][SUP]4,[/SUP][SUP]5[/SUP], Kim JH[SUP]1,[/SUP][SUP]2,[/SUP][SUP]3,[/SUP][SUP]4,[/SUP][SUP]5[/SUP].
[h=3]Author information[/h]
[h=3]Abstract[/h] 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.
[h=4]KEYWORDS:[/h] Generic primer; influenza A virus; matrix gene; real-time PCR; ribonucleoprotein
PMID: 30795722 DOI: 10.1177/1040638719830760