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. 1995 May;33(5):1180–1184. doi: 10.1128/jcm.33.5.1180-1184.1995

Typing and subtyping of influenza viruses in clinical samples by PCR.

K E Wright 1, G A Wilson 1, D Novosad 1, C Dimock 1, D Tan 1, J M Weber 1
PMCID: PMC228127  PMID: 7615726

Abstract

Type A and B influenza viruses can cause a wide spectrum of illness, and these viruses are responsible for considerable mortality and morbidity. Rapid typing of isolates is desirable when amantadine treatment or prophylaxis of contacts of type A influenza virus carriers is considered, but the available rapid techniques lack sensitivity and standard diagnostic methods require expansion of virus in tissue culture or embryonated hens' eggs. We developed a series of oligonucleotide primers able to detect, type, and subtype type A influenza viruses in a single reverse transcription-PCR. RNA was isolated from clinical specimens, and cDNA was generated with random primers. PCR was carried out with a mixture of primers specific for influenza viruses of types B, A/H1 and A/H3, and subtyping of the neuraminidase was carried out on the same cDNA template under identical conditions. Amplified products were detected by ethidium bromide staining of amplified products after agarose gel electrophoresis. When it was used to test 98 clinical specimens, this method was comparable to standard culture techniques in the detection, typing, and subtyping of influenza viruses.

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Selected References

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  1. Air G. M. Sequence relationships among the hemagglutinin genes of 12 subtypes of influenza A virus. Proc Natl Acad Sci U S A. 1981 Dec;78(12):7639–7643. doi: 10.1073/pnas.78.12.7639. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Bao-Lan L., Webster R. G., Brown L. E., Nerome K. Heterogeneity of influenza B viruses. Bull World Health Organ. 1983;61(4):681–687. [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 Mar 16;167(2):425–430. doi: 10.1016/0006-291x(90)92040-7. [DOI] [PubMed] [Google Scholar]
  4. Bucher D. J., Mikhail A., Popple S., Graves P., Meiklejohn G., Hodes D. S., Johansson K., Halonen P. E. Rapid detection of type A influenza viruses with monoclonal antibodies to the M protein (M1) by enzyme-linked immunosorbent assay and time-resolved fluoroimmunoassay. J Clin Microbiol. 1991 Nov;29(11):2484–2488. doi: 10.1128/jcm.29.11.2484-2488.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Cherian T., Bobo L., Steinhoff M. C., Karron R. A., Yolken R. H. Use of PCR-enzyme immunoassay for identification of influenza A virus matrix RNA in clinical samples negative for cultivable virus. J Clin Microbiol. 1994 Mar;32(3):623–628. doi: 10.1128/jcm.32.3.623-628.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Chomel J. J., Remilleux M. F., Marchand P., Aymard M. Rapid diagnosis of influenza A. Comparison with ELISA immunocapture and culture. J Virol Methods. 1992 Jun;37(3):337–343. doi: 10.1016/0166-0934(92)90033-a. [DOI] [PubMed] [Google Scholar]
  7. Claas E. C., Sprenger M. J., Kleter G. E., van Beek R., Quint W. G., Masurel N. Type-specific identification of influenza viruses A, B and C by the polymerase chain reaction. J Virol Methods. 1992 Sep;39(1-2):1–13. doi: 10.1016/0166-0934(92)90120-3. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Claas E. C., van Milaan A. J., Sprenger M. J., Ruiten-Stuiver M., Arron G. I., Rothbarth P. H., Masurel N. Prospective application of reverse transcriptase polymerase chain reaction for diagnosing influenza infections in respiratory samples from a children's hospital. J Clin Microbiol. 1993 Aug;31(8):2218–2221. doi: 10.1128/jcm.31.8.2218-2221.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dickover R. E., Donovan R. M., Goldstein E., Dandekar S., Bush C. E., Carlson J. R. Quantitation of human immunodeficiency virus DNA by using the polymerase chain reaction. J Clin Microbiol. 1990 Sep;28(9):2130–2133. doi: 10.1128/jcm.28.9.2130-2133.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Dominguez E. A., Taber L. H., Couch R. B. Comparison of rapid diagnostic techniques for respiratory syncytial and influenza A virus respiratory infections in young children. J Clin Microbiol. 1993 Sep;31(9):2286–2290. doi: 10.1128/jcm.31.9.2286-2290.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Eldadah Z. A., Asher D. M., Godec M. S., Pomeroy K. L., Goldfarb L. G., Feinstone S. M., Levitan H., Gibbs C. J., Jr, Gajdusek D. C. Detection of flaviviruses by reverse-transcriptase polymerase chain reaction. J Med Virol. 1991 Apr;33(4):260–267. doi: 10.1002/jmv.1890330410. [DOI] [PubMed] [Google Scholar]
  12. Gouvea V., Allen J. R., Glass R. I., Fang Z. Y., Bremont M., Cohen J., McCrae M. A., Saif L. J., Sinarachatanant P., Caul E. O. Detection of group B and C rotaviruses by polymerase chain reaction. J Clin Microbiol. 1991 Mar;29(3):519–523. doi: 10.1128/jcm.29.3.519-523.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Gouvea V., Glass R. I., Woods P., Taniguchi K., Clark H. F., Forrester B., Fang Z. Y. Polymerase chain reaction amplification and typing of rotavirus nucleic acid from stool specimens. J Clin Microbiol. 1990 Feb;28(2):276–282. doi: 10.1128/jcm.28.2.276-282.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Johnston S. L., Bloy H. Evaluation of a rapid enzyme immunoassay for detection of influenza A virus. J Clin Microbiol. 1993 Jan;31(1):142–143. doi: 10.1128/jcm.31.1.142-143.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Larzul D., Guigue F., Sninsky J. J., Mack D. H., Bréchot C., Guesdon J. L. Detection of hepatitis B virus sequences in serum by using in vitro enzymatic amplification. J Virol Methods. 1988 Jul;20(3):227–237. doi: 10.1016/0166-0934(88)90126-7. [DOI] [PubMed] [Google Scholar]
  16. Lozano M. E., Ghiringhelli P. D., Romanowski V., Grau O. A simple nucleic acid amplification assay for the rapid detection of Junín virus in whole blood samples. Virus Res. 1993 Jan;27(1):37–53. doi: 10.1016/0168-1702(93)90111-y. [DOI] [PubMed] [Google Scholar]
  17. Lunkenheimer K., Hufert F. T., Schmitz H. Detection of Lassa virus RNA in specimens from patients with Lassa fever by using the polymerase chain reaction. J Clin Microbiol. 1990 Dec;28(12):2689–2692. doi: 10.1128/jcm.28.12.2689-2692.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Miller H. R., Phipps P. H., Rossier E. Reduction of nonspecific fluorescence in respiratory specimens by pretreatment with N-acetylcysteine. J Clin Microbiol. 1986 Sep;24(3):470–471. doi: 10.1128/jcm.24.3.470-471.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Pisareva M., Bechtereva T., Plyusnin A., Dobretsova A., Kisselev O. PCR-amplification of influenza A virus specific sequences. Arch Virol. 1992;125(1-4):313–318. doi: 10.1007/BF01309648. [DOI] [PubMed] [Google Scholar]
  20. Sellner L. N., Coelen R. J., Mackenzie J. S. A one-tube, one manipulation RT-PCR reaction for detection of Ross River virus. J Virol Methods. 1992 Dec 1;40(3):255–263. doi: 10.1016/0166-0934(92)90084-q. [DOI] [PubMed] [Google Scholar]
  21. Shibata D. K., Arnheim N., Martin W. J. Detection of human papilloma virus in paraffin-embedded tissue using the polymerase chain reaction. J Exp Med. 1988 Jan 1;167(1):225–230. doi: 10.1084/jem.167.1.225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Waner J. L., Todd S. J., Shalaby H., Murphy P., Wall L. V. Comparison of Directigen FLU-A with viral isolation and direct immunofluorescence for the rapid detection and identification of influenza A virus. J Clin Microbiol. 1991 Mar;29(3):479–482. doi: 10.1128/jcm.29.3.479-482.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. 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(3):259–265. doi: 10.1111/j.1348-0421.1991.tb01555.x. [DOI] [PubMed] [Google Scholar]
  24. Zhang W. D., Evans D. H. Detection and identification of human influenza viruses by the polymerase chain reaction. J Virol Methods. 1991 Jun;33(1-2):165–189. doi: 10.1016/0166-0934(91)90017-t. [DOI] [PubMed] [Google Scholar]

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