Type-specific identification of influenza viruses A, B and C by the polymerase chain reaction (original) (raw)

Abstract

The aim of this study was to develop a polymerase chain reaction for specific detection of influenza A, B, and C RNA genomes. Three primer sets were selected from conserved regions of the genome coding for the non-structural proteins and were tested on 61 influenza A (22 H1N1, 9 H2N2, and 30 H3N2), 11 influenza B, and three influenza C isolates. Specific amplified products were obtained with all these strains after electrophoresis on a 2% agarose gel. The specificity of the reaction was increased by hybridization with oligonucleotide probes. When nucleic acids from a variety of micro-organisms from the respiratory tract were subjected to the PCR with these primers, no specific amplified products were generated. The sensitivity of the technique was found to be at the subpicogram level. The RNA-PCR was applied to 21 clinical specimens from patients with a culture/IF proven influenza infection. Six influenza A positive patients and 13 influenza B positive patients could be confirmed in the RNA-PCR. In two cases, influenza B positive IF specimens were found negative by the PCR. No virus could be isolated on eggs or tissue culture from these samples. RNA-PCR is a specific and sensitive technique for the detection of influenza virus genomes.

Keywords: Influenza, Non-structural, PCR, Clinical specimen, Diagnosis

References

  1. Air G.M., Laver W.G. Influenza viruses. In: van Regenmortel M.H.V., Neurath A.R., editors. Immunochemistry of Viruses, II. The basis for serodiagnosis and vaccines. Elsevier; Amsterdam: 1990. pp. 171–216. [Google Scholar]
  2. Bernet C., Garret M., De Barbeyrac B., Bebear C., Bonnet J. Detection of Mycoplasma pneumoniae by using the polymerase chain reaction. J. Clin. Microbiol. 1989;27:2492–2496. doi: 10.1128/jcm.27.11.2492-2496.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bressoud A., Whitcomb J., Pourzand C., Haller O., Cerutti P. Rapid detection of influenza virus H1 by the polymerase chain reaction. Biochem. Biophys. Res. Commun. 1990;167:425–430. doi: 10.1016/0006-291x(90)92040-7. [DOI] [PubMed] [Google Scholar]
  4. Buonagurio D.A., Nakada S., Parvin J.D., Krystal M., Palese P., Fitch W.M. Evolution of human influenza A viruses over 50 years: rapid, uniform rate of change in NS gene. Science. 1986;232:980–982. doi: 10.1126/science.2939560. [DOI] [PubMed] [Google Scholar]
  5. Buonagurio D.A., Nakada S., Fitch W.M., Palese P. Epidemiology of influenza C in man: multiple evolutionary lineages and low rate of change. Virology. 1986;153:12–21. doi: 10.1016/0042-6822(86)90003-6. [DOI] [PubMed] [Google Scholar]
  6. Chomczynski P., Sacchi N. Single step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem. 1987;162:156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  7. Claas H.C.J., Wagenvoort J.H.T., Niesters H.G.M., Tio T.T., Van Rijsoort-Vos J.H., Quint W.G.V. Diagnostic value of the polymerase chain reaction for Chlamydia detection as determined in a follow-up study. J. Clin. Microbiol. 1991;29:42–45. doi: 10.1128/jcm.29.1.42-45.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Engblom E., Ekfors T.O., Meurman O.H., Toivanen A., Nikoskelainen Fatal influenza A myocarditis with isolation of virus from myocardium. Acta Med. Scand. 1983;213:75–78. doi: 10.1111/j.0954-6820.1983.tb03693.x. [DOI] [PubMed] [Google Scholar]
  9. Espy M.J., Schmidt T.F., Harmon M.W., Kendall A.P. Rapid detection of influenza virus by shell vial assay with monoclonal antibodies. J. Clin. Microbiol. 1986;24:677–679. doi: 10.1128/jcm.24.4.677-679.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Katz J.M., Wang M., Webster R.G. Direct sequencing of the HA gene of influenza (H3N2) virus in original clinical samples reveals sequence identity with mammalian cell-grown virus. J. Virol. 1990;64:1808–1811. doi: 10.1128/jvi.64.4.1808-1811.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Kwok S., Higuchi R. Avoiding false positives with PCR. Nature. 1989;339:237–238. doi: 10.1038/339237a0. [DOI] [PubMed] [Google Scholar]
  12. Lamb R.A. Genes and proteins of the influenza viruses. In: Krug R.M., editor. The influenza viruses. Plenum Press; New York: 1989. pp. 1–87. [Google Scholar]
  13. Ludwig S., Schultz U., Mandler J., Fitch W.M., Scholtissek C. Phylogenetic relationship of the nonstructural (NS) genes of influenza A viruses. Virology. 1991;183:566–577. doi: 10.1016/0042-6822(91)90985-k. [DOI] [PubMed] [Google Scholar]
  14. Madin S.H., Darby N.B. Vol. 98. 1958. Established kidney cell lines of normal adult bovine and ovine origin; pp. 574–576. (Proc. Soc. Exp. Biol. Med.). [DOI] [PubMed] [Google Scholar]
  15. Oseasohn R., Adelson L., Masaro K. Clinopathologic study of thirty-three fatal cases of Asian influenza. N. Engl. J. Med. 1959;260:509–518. doi: 10.1056/NEJM195903122601101. [DOI] [PubMed] [Google Scholar]
  16. Queen C., Korn L.J. A comprehensive sequence analysis program for the IBM personal computer. Nucleic Acids Res. 1984;12:581–599. doi: 10.1093/nar/12.1part2.581. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Rajakumar A., Swierkosz E.M., Schulze I.T. Vol. 87. 1990. Sequence of an influenza virus hemagglutinin determined directly from a clinical sample; pp. 4154–4158. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Robertson J.S., Bootman J.S., Nicolson C., Major D., Robertson E.W., Wood J.M. The hemagglutinin of influenza B virus present in clinical material is a single species identical to that of mammalian cell-grown virus. Virology. 1990;179:35–40. doi: 10.1016/0042-6822(90)90270-2. [DOI] [PubMed] [Google Scholar]
  19. Sambrook J., Fritsch E.F., Maniatis T. 2nd edn. Cold Spring Harbor Laboratory; Cold Spring Harbor, NY: 1989. Molecular cloning: a laboratory manual. [Google Scholar]
  20. Sprenger M.J., Diepersloot R.J., Beyer W.E., Masurel N. Influenza-related excess mortality in the Netherlands 1989/1990. Lancet. 1990;ii:382. doi: 10.1016/0140-6736(90)91929-5. [DOI] [PubMed] [Google Scholar]
  21. Stokes C.E., Bernstein J.M., Kyger S.A., Hayden F.G. Rapid diagnosis of influenza A and B by 24-h fluorescent focus assay. J. Clin. Microbiol. 1988;26:1263–1266. doi: 10.1128/jcm.26.7.1263-1266.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Tillett H.E., Smith J.W.G., Gooch C.D. Excess deaths attributable to influenza in England and Wales: age at death and certified cause. Int. J. Epidemiol. 1983;12:344–352. doi: 10.1093/ije/12.3.344. [DOI] [PubMed] [Google Scholar]
  23. Wright P.A., Wynford-Thomas D. The polymerase chain reaction: miracle or mirage? A critical review of its uses and limitations in diagnosis and research. J. Pathol. 1990;162:99–117. doi: 10.1002/path.1711620203. [DOI] [PubMed] [Google Scholar]
  24. Yamada A., Imanishi J., Nakajima E., Nakajima K., Nakajima S. Detection of influenza viruses in throat swab by using polymerase chain reaction. Microbiol. Immunol. 1991;35:259–265. doi: 10.1111/j.1348-0421.1991.tb01555.x. [DOI] [PubMed] [Google Scholar]
  25. Yamashita M., Krystal M., Fitch W.M., Palese P. Influenza B virus evolution: cocirculating lineages and comparison of evolutionary pattern with those of influenza A and C viruses. Virology. 1988;163:112–122. doi: 10.1016/0042-6822(88)90238-3. [DOI] [PubMed] [Google Scholar]
  26. Zhang W., Evans D.H. Detection and identification of human influenza viruses by the polymerase chain reaction. J. Virol. Meth. 1991;33:165–189. doi: 10.1016/0166-0934(91)90017-t. [DOI] [PubMed] [Google Scholar]