Skip to main content
PMC home page
Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 1994 Feb;32(2):285–291. doi: 10.1128/jcm.32.2.285-291.1994

Nested PCR for specific detection and rapid identification of human picornaviruses.

U Kämmerer 1, B Kunkel 1, K Korn 1
PMCID: PMC263026  PMID: 8150936

Abstract

A nested PCR for the detection and rapid identification of human picornaviruses is described. Enteroviruses and rhinoviruses were amplified with the same set of four primers from the 5'-noncoding region. The nested primers allowed the detection of far less than 1 PFU in diluted virus stocks without Southern blot hybridization. In patients with neurological disorders (mainly aseptic meningitis), 43% of 37 specimens (11 of 21 cerebrospinal fluid specimens, 2 of 10 serum specimens, and 3 of 6 stool specimens) were positive by PCR. A total of 21% (10 of 47 specimens) of heart biopsy specimens from patients with dilative cardiomyopathy were PCR positive, whereas 3% (2 of 70 specimens) of control biopsy specimens from patients with coronary artery disease were PCR positive. PCR-amplified fragments from 27 of 29 clinical isolates and 14 of 28 patient samples were successfully serotyped by restriction enzyme digestion. Two specimens were further investigated by direct sequencing of PCR products, leading to the identification of a poliovirus type 3 isolate with a sequence that was highly divergent from previously published sequences.

Full text

PDF
285

Images in this article

287Image
on p.287
287Image
on p.287

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Chang K. H., Auvinen P., Hyypiä T., Stanway G. The nucleotide sequence of coxsackievirus A9; implications for receptor binding and enterovirus classification. J Gen Virol. 1989 Dec;70(Pt 12):3269–3280. doi: 10.1099/0022-1317-70-12-3269. [DOI] [PubMed] [Google Scholar]
  2. 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]
  3. Chomczynski P., Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem. 1987 Apr;162(1):156–159. doi: 10.1006/abio.1987.9999. [DOI] [PubMed] [Google Scholar]
  4. Couch R. B. The common cold: control? J Infect Dis. 1984 Aug;150(2):167–173. doi: 10.1093/infdis/150.2.167. [DOI] [PubMed] [Google Scholar]
  5. Duechler M., Skern T., Sommergruber W., Neubauer C., Gruendler P., Fogy I., Blaas D., Kuechler E. Evolutionary relationships within the human rhinovirus genus: comparison of serotypes 89, 2, and 14. Proc Natl Acad Sci U S A. 1987 May;84(9):2605–2609. doi: 10.1073/pnas.84.9.2605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. 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]
  7. 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]
  8. Hughes P. J., Evans D. M., Minor P. D., Schild G. C., Almond J. W., Stanway G. The nucleotide sequence of a type 3 poliovirus isolated during a recent outbreak of poliomyelitis in Finland. J Gen Virol. 1986 Oct;67(Pt 10):2093–2102. doi: 10.1099/0022-1317-67-10-2093. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Hughes P. J., North C., Minor P. D., Stanway G. The complete nucleotide sequence of coxsackievirus A21. J Gen Virol. 1989 Nov;70(Pt 11):2943–2952. doi: 10.1099/0022-1317-70-11-2943. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Iizuka N., Kuge S., Nomoto A. Complete nucleotide sequence of the genome of coxsackievirus B1. Virology. 1987 Jan;156(1):64–73. doi: 10.1016/0042-6822(87)90436-3. [DOI] [PubMed] [Google Scholar]
  13. Inoue T., Suzuki T., Sekiguchi K. The complete nucleotide sequence of swine vesicular disease virus. J Gen Virol. 1989 Apr;70(Pt 4):919–934. doi: 10.1099/0022-1317-70-4-919. [DOI] [PubMed] [Google Scholar]
  14. Jenkins O., Booth J. D., Minor P. D., Almond J. W. The complete nucleotide sequence of coxsackievirus B4 and its comparison to other members of the Picornaviridae. J Gen Virol. 1987 Jul;68(Pt 7):1835–1848. doi: 10.1099/0022-1317-68-7-1835. [DOI] [PubMed] [Google Scholar]
  15. Jin O., Sole M. J., Butany J. W., Chia W. K., McLaughlin P. R., Liu P., Liew C. C. Detection of enterovirus RNA in myocardial biopsies from patients with myocarditis and cardiomyopathy using gene amplification by polymerase chain reaction. Circulation. 1990 Jul;82(1):8–16. doi: 10.1161/01.cir.82.1.8. [DOI] [PubMed] [Google Scholar]
  16. 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]
  17. Kellner G., Popow-Kraupp T., Kundi M., Binder C., Wallner H., Kunz C. Contribution of rhinoviruses to respiratory viral infections in childhood: a prospective study in a mainly hospitalized infant population. J Med Virol. 1988 Aug;25(4):455–469. doi: 10.1002/jmv.1890250409. [DOI] [PubMed] [Google Scholar]
  18. Krilov L., Pierik L., Keller E., Mahan K., Watson D., Hirsch M., Hamparian V., McIntosh K. The association of rhinoviruses with lower respiratory tract disease in hospitalized patients. J Med Virol. 1986 Aug;19(4):345–352. doi: 10.1002/jmv.1890190407. [DOI] [PubMed] [Google Scholar]
  19. Kwok S., Higuchi R. Avoiding false positives with PCR. Nature. 1989 May 18;339(6221):237–238. doi: 10.1038/339237a0. [DOI] [PubMed] [Google Scholar]
  20. Lindberg A. M., Stålhandske P. O., Pettersson U. Genome of coxsackievirus B3. Virology. 1987 Jan;156(1):50–63. doi: 10.1016/0042-6822(87)90435-1. [DOI] [PubMed] [Google Scholar]
  21. Minor P. D. The molecular biology of poliovaccines. J Gen Virol. 1992 Dec;73(Pt 12):3065–3077. doi: 10.1099/0022-1317-73-12-3065. [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. Nomoto A., Omata T., Toyoda H., Kuge S., Horie H., Kataoka Y., Genba Y., Nakano Y., Imura N. Complete nucleotide sequence of the attenuated poliovirus Sabin 1 strain genome. Proc Natl Acad Sci U S A. 1982 Oct;79(19):5793–5797. doi: 10.1073/pnas.79.19.5793. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. 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]
  25. 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]
  26. 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]
  27. Rivera V. M., Welsh J. D., Maizel J. V., Jr Comparative sequence analysis of the 5' noncoding region of the enteroviruses and rhinoviruses. Virology. 1988 Jul;165(1):42–50. doi: 10.1016/0042-6822(88)90656-3. [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., Kinsella J. P., Wasserman R. L. Persistent enterovirus infection in culture-negative meningoencephalitis: demonstration by enzymatic RNA amplification. J Infect Dis. 1990 Apr;161(4):787–791. doi: 10.1093/infdis/161.4.787. [DOI] [PubMed] [Google Scholar]
  30. Ryan M. D., Jenkins O., Hughes P. J., Brown A., Knowles N. J., Booth D., Minor P. D., Almond J. W. The complete nucleotide sequence of enterovirus type 70: relationships with other members of the picornaviridae. J Gen Virol. 1990 Oct;71(Pt 10):2291–2299. doi: 10.1099/0022-1317-71-10-2291. [DOI] [PubMed] [Google Scholar]
  31. Saiki R. K., Gelfand D. H., Stoffel S., Scharf S. J., Higuchi R., Horn G. T., Mullis K. B., Erlich H. A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988 Jan 29;239(4839):487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  32. Severini G. M., Mestroni L., Falaschi A., Camerini F., Giacca M. Nested polymerase chain reaction for high-sensitivity detection of enteroviral RNA in biological samples. J Clin Microbiol. 1993 May;31(5):1345–1349. doi: 10.1128/jcm.31.5.1345-1349.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Skern T., Sommergruber W., Blaas D., Gruendler P., Fraundorfer F., Pieler C., Fogy I., Kuechler E. Human rhinovirus 2: complete nucleotide sequence and proteolytic processing signals in the capsid protein region. Nucleic Acids Res. 1985 Mar 25;13(6):2111–2126. doi: 10.1093/nar/13.6.2111. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Stanway G., Hughes P. J., Mountford R. C., Minor P. D., Almond J. W. The complete nucleotide sequence of a common cold virus: human rhinovirus 14. Nucleic Acids Res. 1984 Oct 25;12(20):7859–7875. doi: 10.1093/nar/12.20.7859. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Stanway G., Hughes P. J., Mountford R. C., Reeve P., Minor P. D., Schild G. C., Almond J. W. Comparison of the complete nucleotide sequences of the genomes of the neurovirulent poliovirus P3/Leon/37 and its attenuated Sabin vaccine derivative P3/Leon 12a1b. Proc Natl Acad Sci U S A. 1984 Mar;81(5):1539–1543. doi: 10.1073/pnas.81.5.1539. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. 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]
  37. Supanaranond K., Takeda N., Yamazaki S. The complete nucleotide sequence of a variant of Coxsackievirus A24, an agent causing acute hemorrhagic conjunctivitis. Virus Genes. 1992 Apr;6(2):149–158. doi: 10.1007/BF01703064. [DOI] [PubMed] [Google Scholar]
  38. 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]
  39. Toyoda H., Kohara M., Kataoka Y., Suganuma T., Omata T., Imura N., Nomoto A. Complete nucleotide sequences of all three poliovirus serotype genomes. Implication for genetic relationship, gene function and antigenic determinants. J Mol Biol. 1984 Apr 25;174(4):561–585. doi: 10.1016/0022-2836(84)90084-6. [DOI] [PubMed] [Google Scholar]
  40. 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]

Articles from Journal of Clinical Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES