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

Journal of Veterinary Diagnostic Investigation : Official Publication of the American Association of Veterinary Laboratory Diagnosticians, Inc
Ji-Woon KimJae-Hong 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.

References

Jan 1, 1977·Archives of Virology·J T HeywardJ F Obijeski
Jan 1, 1985·Analytical Biochemistry·D J AroraS Boileau
Dec 1, 1993·PCR Methods and Applications·C W DieffenbachG S Dveksler
May 10, 2000·Proceedings of the National Academy of Sciences of the United States of America·E HoffmannR G Webster
Jun 22, 2000·Journal of Veterinary Medicine. B, Infectious Diseases and Veterinary Public Health·E StarickO Werner
Aug 10, 2002·Bioinformatics·Chaim Linhart, Ron Shamir
Dec 20, 2003·Biochemical and Biophysical Research Communications·Lok-Ting LauAlbert C H Yu
Feb 15, 2005·Journal of Virology·Ron A M FouchierAlbert D M E Osterhaus
May 11, 2005·Journal of Computational Biology : a Journal of Computational Molecular Cell Biology·Chaim Linhart, Ron Shamir
Jul 21, 2005·Proceedings of the National Academy of Sciences of the United States of America·D J Hulse-PostR G Webster
Apr 18, 2008·Nature·Andrew RambautEdward C Holmes
Dec 2, 2009·The Journal of Molecular Diagnostics : JMD·Ralph StadhoudersMartin Schutten
Jul 24, 2012·Nucleic Acids Research·Mark B SteadSidney R Kushner
Feb 24, 2016·Diagnostic Microbiology and Infectious Disease·J C Nguyen VanA Le Monnier
Sep 2, 2016·Journal of Clinical Microbiology·Frederick S NolteSusan B Barrett
Dec 3, 2016·Journal of Clinical Virology : the Official Publication of the Pan American Society for Clinical Virology·Francesca ColavitaConcetta Castilletti

❮ Previous
Next ❯

Methods Mentioned

BETA
electrophoresis
PCR

Software Mentioned

StepOne
Excel

Related Concepts

Related Feeds

Antivirals

Antivirals are medications that are used specifically for treating viral infections. Discover the latest research on antivirals here.

Antivirals (ASM)

Antivirals are medications that are used specifically for treating viral infections. Discover the latest research on antivirals here.