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J Vet Diagn Invest. 2019 Mar;31(2):175-183. doi: 10.1177/1040638719830760. Epub 2019 Feb 22.

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

Kim JW1,2,3,4,5, Lee CY1,2,3,4,5, Nguyen TT1,2,3,4,5, Kim IH1,2,3,4,5, Kwon HJ1,2,3,4,5, Kim JH1,2,3,4,5.

Author information

1
Laboratory of Avian Diseases (J-W Kim, Lee, Nguyen, J-H Kim), College of Veterinary Medicine, Seoul National University, Seoul, Republic of Korea.
2
Department of Farm Animal Medicine (Kwon), College of Veterinary Medicine, Seoul National University, Seoul, Republic of Korea.
3
Farm Animal Clinical Training and Research Center, Institutes of Green Bio Science and Technology (Kwon), College of Veterinary Medicine, Seoul National University, Seoul, Republic of Korea.
4
Research Institute for Veterinary Science (Kwon, J-H Kim), College of Veterinary Medicine, Seoul National University, Seoul, Republic of Korea.
5
Division of Antimicrobial Resistance, Center for Infectious Diseases, National Research Institute of Health, Cheongju, Republic of Korea (I-H Kim).

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.

KEYWORDS:

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

PMID:
30795722
PMCID:
PMC6838833
[Available on 2020-02-22]
DOI:
10.1177/1040638719830760
[Indexed for MEDLINE]

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