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. 1995 Mar;33(3):648–653. doi: 10.1128/jcm.33.3.648-653.1995

Detection of enteroviruses and rhinoviruses in clinical specimens by PCR and liquid-phase hybridization.

P Halonen 1, E Rocha 1, J Hierholzer 1, B Holloway 1, T Hyypiä 1, P Hurskainen 1, M Pallansch 1
PMCID: PMC228007  PMID: 7751371

Abstract

A sensitive method based on PCR followed by liquid-phase hybridization for detection of enterovirus and rhinovirus RNAs in clinical specimens and cell culture supernatants is described. RNA was extracted from stool samples, throat swabs, nasopharyngeal aspirates, cerebrospinal fluid, urine, and plasma with a commercial phenol-guanidinium-chloroform reagent and purified on a polysulfone membrane, on which the reverse transcriptase reaction was also done. Two sets of oligonucleotide primers from the 5' noncoding region of picornaviruses were selected for DNA amplification of 153-bp (enterovirus) and 120-bp (rhinovirus) regions. Double-stranded amplicons were digested into single strands with T7 gene 6 exonuclease and quantitated by an assay using a europium-labeled probe, streptavidin- and biotinylated probe-coated microtitration wells, and time-resolved fluorometry. The sensitivity of the assay was about one template molecule when purified coxsackievirus A9 RNA was used. All enterovirus prototype strains, except echoviruses 22 and 23, and clinical isolates grown in cell culture or suckling mice were strongly positive by the enterovirus PCR-hybridization, as were selected prototype strains and untyped isolates of rhinoviruses by the rhinovirus PCR-hybridization. In a series of 100 clinical specimens tested, the results for 92 agreed with virus culture results. The detection method described will be useful in etiopathogenic studies on enteroviruses and rhinoviruses.

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Selected References

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  1. Abebe A., Johansson B., Abens J., Strannegård O. Detection of enteroviruses in faeces by polymerase chain reaction. Scand J Infect Dis. 1992;24(3):265–273. doi: 10.3109/00365549209061331. [DOI] [PubMed] [Google Scholar]
  2. Arola M., Ziegler T., Ruuskanen O., Mertsola J., Näntö-Salonen K., Halonen P. Rhinovirus in acute otitis media. J Pediatr. 1988 Oct;113(4):693–695. doi: 10.1016/S0022-3476(88)80380-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Arruda E., Hayden F. G. Detection of human rhinovirus RNA in nasal washings by PCR. Mol Cell Probes. 1993 Oct;7(5):373–379. doi: 10.1006/mcpr.1993.1055. [DOI] [PubMed] [Google Scholar]
  4. Chapman N. M., Tracy S., Gauntt C. J., Fortmueller U. Molecular detection and identification of enteroviruses using enzymatic amplification and nucleic acid hybridization. J Clin Microbiol. 1990 May;28(5):843–850. doi: 10.1128/jcm.28.5.843-850.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Coller B. A., Chapman N. M., Beck M. A., Pallansch M. A., Gauntt C. J., Tracy S. M. Echovirus 22 is an atypical enterovirus. J Virol. 1990 Jun;64(6):2692–2701. doi: 10.1128/jvi.64.6.2692-2701.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Dahlén P. O., Iitiä A. J., Skagius G., Frostell A., Nunn M. F., Kwiatkowski M. Detection of human immunodeficiency virus type 1 by using the polymerase chain reaction and a time-resolved fluorescence-based hybridization assay. J Clin Microbiol. 1991 Apr;29(4):798–804. doi: 10.1128/jcm.29.4.798-804.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dahlén P. Detection of biotinylated DNA probes by using Eu-labeled streptavidin and time-resolved fluorometry. Anal Biochem. 1987 Jul;164(1):78–83. doi: 10.1016/0003-2697(87)90370-8. [DOI] [PubMed] [Google Scholar]
  8. Gama R. E., Horsnell P. R., Hughes P. J., North C., Bruce C. B., al-Nakib W., Stanway G. Amplification of rhinovirus specific nucleic acids from clinical samples using the polymerase chain reaction. J Med Virol. 1989 Jun;28(2):73–77. doi: 10.1002/jmv.1890280204. [DOI] [PubMed] [Google Scholar]
  9. Gama R. E., Hughes P. J., Bruce C. B., Stanway G. Polymerase chain reaction amplification of rhinovirus nucleic acids from clinical material. Nucleic Acids Res. 1988 Oct 11;16(19):9346–9346. doi: 10.1093/nar/16.19.9346. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hierholzer J. C., Halonen P. E., Dahlen P. O., Bingham P. G., McDonough M. M. Detection of adenovirus in clinical specimens by polymerase chain reaction and liquid-phase hybridization quantitated by time-resolved fluorometry. J Clin Microbiol. 1993 Jul;31(7):1886–1891. doi: 10.1128/jcm.31.7.1886-1891.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Holloway B., Erdman D. D., Durigon E. L., Murtagh J. J., Jr An exonuclease-amplification coupled capture technique improves detection of PCR product. Nucleic Acids Res. 1993 Aug 11;21(16):3905–3906. doi: 10.1093/nar/21.16.3905. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Hughes P. J., North C., Jellis C. H., Minor P. D., Stanway G. The nucleotide sequence of human rhinovirus 1B: molecular relationships within the rhinovirus genus. J Gen Virol. 1988 Jan;69(Pt 1):49–58. doi: 10.1099/0022-1317-69-1-49. [DOI] [PubMed] [Google Scholar]
  13. Hyypiä T., Auvinen P., Maaronen M. Polymerase chain reaction for human picornaviruses. J Gen Virol. 1989 Dec;70(Pt 12):3261–3268. doi: 10.1099/0022-1317-70-12-3261. [DOI] [PubMed] [Google Scholar]
  14. Hyypiä T. Etiological diagnosis of viral heart disease. Scand J Infect Dis Suppl. 1993;88:25–31. [PubMed] [Google Scholar]
  15. Hyypiä T., Horsnell C., Maaronen M., Khan M., Kalkkinen N., Auvinen P., Kinnunen L., Stanway G. A distinct picornavirus group identified by sequence analysis. Proc Natl Acad Sci U S A. 1992 Sep 15;89(18):8847–8851. doi: 10.1073/pnas.89.18.8847. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ireland D. C., Kent J., Nicholson K. G. Improved detection of rhinoviruses in nasal and throat swabs by seminested RT-PCR. J Med Virol. 1993 Jun;40(2):96–101. doi: 10.1002/jmv.1890400204. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Johnston S. L., Sanderson G., Pattemore P. K., Smith S., Bardin P. G., Bruce C. B., Lambden P. R., Tyrrell D. A., Holgate S. T. Use of polymerase chain reaction for diagnosis of picornavirus infection in subjects with and without respiratory symptoms. J Clin Microbiol. 1993 Jan;31(1):111–117. doi: 10.1128/jcm.31.1.111-117.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Jongen P. J., Zoll G. J., Beaumont M., Melchers W. J., van de Putte L. B., Galama J. M. Polymyositis and dermatomyositis: no persistence of enterovirus or encephalomyocarditis virus RNA in muscle. Ann Rheum Dis. 1993 Aug;52(8):575–578. doi: 10.1136/ard.52.8.575. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Leff R. L., Love L. A., Miller F. W., Greenberg S. J., Klein E. A., Dalakas M. C., Plotz P. H. Viruses in idiopathic inflammatory myopathies: absence of candidate viral genomes in muscle. Lancet. 1992 May 16;339(8803):1192–1195. doi: 10.1016/0140-6736(92)91134-t. [DOI] [PubMed] [Google Scholar]
  20. Leon-Monzon M., Dalakas M. C. Absence of persistent infection with enteroviruses in muscles of patients with inflammatory myopathies. Ann Neurol. 1992 Aug;32(2):219–222. doi: 10.1002/ana.410320215. [DOI] [PubMed] [Google Scholar]
  21. Liljeqvist J. A., Bergström T., Holmström S., Samuelson A., Yousef G. E., Waagstein F., Jeansson S. Failure to demonstrate enterovirus aetiology in Swedish patients with dilated cardiomyopathy. J Med Virol. 1993 Jan;39(1):6–10. doi: 10.1002/jmv.1890390103. [DOI] [PubMed] [Google Scholar]
  22. Muir P., Nicholson F., Jhetam M., Neogi S., Banatvala J. E. Rapid diagnosis of enterovirus infection by magnetic bead extraction and polymerase chain reaction detection of enterovirus RNA in clinical specimens. J Clin Microbiol. 1993 Jan;31(1):31–38. doi: 10.1128/jcm.31.1.31-38.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  23. Olive D. M., Al-Mufti S., Al-Mulla W., Khan M. A., Pasca A., Stanway G., Al-Nakib W. Detection and differentiation of picornaviruses in clinical samples following genomic amplification. J Gen Virol. 1990 Sep;71(Pt 9):2141–2147. doi: 10.1099/0022-1317-71-9-2141. [DOI] [PubMed] [Google Scholar]
  24. Petitjean J., Kopecka H., Freymuth F., Langlard J. M., Scanu P., Galateau F., Bouhour J. B., Ferrière M., Charbonneau P., Komajda M. Detection of enteroviruses in endomyocardial biopsy by molecular approach. J Med Virol. 1992 May;37(1):76–82. doi: 10.1002/jmv.1890370114. [DOI] [PubMed] [Google Scholar]
  25. Racaniello V. R., Baltimore D. Molecular cloning of poliovirus cDNA and determination of the complete nucleotide sequence of the viral genome. Proc Natl Acad Sci U S A. 1981 Aug;78(8):4887–4891. doi: 10.1073/pnas.78.8.4887. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Ranki M., Palva A., Virtanen M., Laaksonen M., Söderlund H. Sandwich hybridization as a convenient method for the detection of nucleic acids in crude samples. Gene. 1983 Jan-Feb;21(1-2):77–85. doi: 10.1016/0378-1119(83)90149-x. [DOI] [PubMed] [Google Scholar]
  27. Rocha E. P., Xu X., Hall H. E., Allen J. R., Regnery H. L., Cox N. J. Comparison of 10 influenza A (H1N1 and H3N2) haemagglutinin sequences obtained directly from clinical specimens to those of MDCK cell- and egg-grown viruses. J Gen Virol. 1993 Nov;74(Pt 11):2513–2518. doi: 10.1099/0022-1317-74-11-2513. [DOI] [PubMed] [Google Scholar]
  28. Rotbart H. A. Enzymatic RNA amplification of the enteroviruses. J Clin Microbiol. 1990 Mar;28(3):438–442. doi: 10.1128/jcm.28.3.438-442.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Rotbart H. A., Levin M. J., Villarreal L. P. Use of subgenomic poliovirus DNA hybridization probes to detect the major subgroups of enteroviruses. J Clin Microbiol. 1984 Dec;20(6):1105–1108. doi: 10.1128/jcm.20.6.1105-1108.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Sawyer M. H., Holland D., Aintablian N., Connor J. D., Keyser E. F., Waecker N. J., Jr Diagnosis of enteroviral central nervous system infection by polymerase chain reaction during a large community outbreak. Pediatr Infect Dis J. 1994 Mar;13(3):177–182. doi: 10.1097/00006454-199403000-00002. [DOI] [PubMed] [Google Scholar]
  31. Schwaiger A., Umlauft F., Weyrer K., Larcher C., Lyons J., Mühlberger V., Dietze O., Grünewald K. Detection of enteroviral ribonucleic acid in myocardial biopsies from patients with idiopathic dilated cardiomyopathy by polymerase chain reaction. Am Heart J. 1993 Aug;126(2):406–410. doi: 10.1016/0002-8703(93)91058-m. [DOI] [PubMed] [Google Scholar]
  32. Stanway G. Structure, function and evolution of picornaviruses. J Gen Virol. 1990 Nov;71(Pt 11):2483–2501. doi: 10.1099/0022-1317-71-11-2483. [DOI] [PubMed] [Google Scholar]
  33. Syvänen A. C., Laaksonen M., Söderlund H. Fast quantification of nucleic acid hybrids by affinity-based hybrid collection. Nucleic Acids Res. 1986 Jun 25;14(12):5037–5048. doi: 10.1093/nar/14.12.5037. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Thorén A., Robinson A. J., Maguire T., Jenkins R. Two-step PCR in the retrospective diagnosis of enteroviral viraemia. Scand J Infect Dis. 1992;24(2):137–141. doi: 10.3109/00365549209052603. [DOI] [PubMed] [Google Scholar]
  35. Torgersen H., Skern T., Blaas D. Typing of human rhinoviruses based on sequence variations in the 5' non-coding region. J Gen Virol. 1989 Nov;70(Pt 11):3111–3116. doi: 10.1099/0022-1317-70-11-3111. [DOI] [PubMed] [Google Scholar]
  36. Walker G. T., Fraiser M. S., Schram J. L., Little M. C., Nadeau J. G., Malinowski D. P. Strand displacement amplification--an isothermal, in vitro DNA amplification technique. Nucleic Acids Res. 1992 Apr 11;20(7):1691–1696. doi: 10.1093/nar/20.7.1691. [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Yang C. F., De L., Holloway B. P., Pallansch M. A., Kew O. M. Detection and identification of vaccine-related polioviruses by the polymerase chain reaction. Virus Res. 1991 Jul;20(2):159–179. doi: 10.1016/0168-1702(91)90107-7. [DOI] [PubMed] [Google Scholar]
  38. Yang C. F., De L., Yang S. J., Ruiz Gómez J., Cruz J. R., Holloway B. P., Pallansch M. A., Kew O. M. Genotype-specific in vitro amplification of sequences of the wild type 3 polioviruses from Mexico and Guatemala. Virus Res. 1992 Aug;24(3):277–296. doi: 10.1016/0168-1702(92)90124-r. [DOI] [PubMed] [Google Scholar]
  39. Zoll G. J., Melchers W. J., Kopecka H., Jambroes G., van der Poel H. J., Galama J. M. General primer-mediated polymerase chain reaction for detection of enteroviruses: application for diagnostic routine and persistent infections. J Clin Microbiol. 1992 Jan;30(1):160–165. doi: 10.1128/jcm.30.1.160-165.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]

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