Skip to main content
NCBI home page
Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 1991 May;29(5):945–952. doi: 10.1128/jcm.29.5.945-952.1991

Detection of rubella virus gene sequences by enzymatic amplification and direct sequencing of amplified DNA.

F A Eggerding 1, J Peters 1, R K Lee 1, C B Inderlied 1
PMCID: PMC269913  PMID: 2056062

Abstract

We developed a rapid and sensitive polymerase chain reaction (PCR) assay for detecting and identifying rubella virus (RV). A segment of the RV gene which encodes the E1 membrane glycoprotein of RV was selected as a target for PCR amplification. Single-stranded viral RNA, extracted from infected cells or released from virions, was used as a template for reverse transcription followed by PCR amplification with two different sets of primer pairs, one nested within the other. The amplified E1 gene sequences were detected in ethidium bromide-stained agarose minigels, and their identities were verified by restriction enzyme digestion and hybridization to a probe directed at a site within the PCR target. Single-stranded DNA generated by asymmetric amplification of the target was directly sequenced by using fluorescent dideoxy-terminators and an automated procedure in order to confirm the target sequence. This PCR assay provides a rapid confirmatory test for the detection of RV by cell culture and appears to have considerable potential for the direct detection of RV in clinical specimens. The strategy used in the development of this PCR assay should be useful for developing other diagnostic PCR assays for viruses.

Full text

PDF
945

Images in this article

948Image
on p.948
948Image
on p.948
949Image
on p.949
950Image
on p.950

Selected References

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

  1. Brenner C. A., Tam A. W., Nelson P. A., Engleman E. G., Suzuki N., Fry K. E., Larrick J. W. Message amplification phenotyping (MAPPing): a technique to simultaneously measure multiple mRNAs from small numbers of cells. Biotechniques. 1989 Nov-Dec;7(10):1096–1103. [PubMed] [Google Scholar]
  2. Carman W. F., Williamson C., Cunliffe B. A., Kidd A. H. Reverse transcription and subsequent DNA amplification of rubella virus RNA. J Virol Methods. 1989 Jul;25(1):21–29. doi: 10.1016/0166-0934(89)90097-9. [DOI] [PubMed] [Google Scholar]
  3. Chaconas G., van de Sande J. H. 5'-32P labeling of RNA and DNA restriction fragments. Methods Enzymol. 1980;65(1):75–85. doi: 10.1016/s0076-6879(80)65012-5. [DOI] [PubMed] [Google Scholar]
  4. Chantler J. K., Ford D. K., Tingle A. J. Persistent rubella infection and rubella-associated arthritis. Lancet. 1982 Jun 12;1(8285):1323–1325. doi: 10.1016/s0140-6736(82)92398-4. [DOI] [PubMed] [Google Scholar]
  5. Curiel D. T., Buchhagen D. L., Chiba I., D'Amico D., Takahashi T., Minna J. D. A chemical mismatch cleavage method useful for the detection of point mutations in the p53 gene in lung cancer. Am J Respir Cell Mol Biol. 1990 Nov;3(5):405–411. doi: 10.1165/ajrcmb/3.5.405. [DOI] [PubMed] [Google Scholar]
  6. Eggerding F. A. Alterations in early adenovirus transcription and mRNA abundance induced by translational inhibitors. J Biol Chem. 1985 Jul 15;260(14):8354–8359. [PubMed] [Google Scholar]
  7. Filipenko D., Hobman T., MacDonald I., Gillam S. In situ detection of rubella RNA and antigens in cultured cells. J Virol Methods. 1988 Oct;22(1):109–118. doi: 10.1016/0166-0934(88)90093-6. [DOI] [PubMed] [Google Scholar]
  8. Frey T. K., Marr L. D., Hemphill M. L., Dominguez G. Molecular cloning and sequencing of the region of the rubella virus genome coding for glycoprotein E1. Virology. 1986 Oct 15;154(1):228–232. doi: 10.1016/0042-6822(86)90446-0. [DOI] [PubMed] [Google Scholar]
  9. Gyllensten U. B., Erlich H. A. Generation of single-stranded DNA by the polymerase chain reaction and its application to direct sequencing of the HLA-DQA locus. Proc Natl Acad Sci U S A. 1988 Oct;85(20):7652–7656. doi: 10.1073/pnas.85.20.7652. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Hemphill M. L., Forng R. Y., Abernathy E. S., Frey T. K. Time course of virus-specific macromolecular synthesis during rubella virus infection in Vero cells. Virology. 1988 Jan;162(1):65–75. doi: 10.1016/0042-6822(88)90395-9. [DOI] [PubMed] [Google Scholar]
  11. Ho-Terry L., Terry G. M., Londesborough P. Diagnosis of foetal rubella virus infection by polymerase chain reaction. J Gen Virol. 1990 Jul;71(Pt 7):1607–1611. doi: 10.1099/0022-1317-71-7-1607. [DOI] [PubMed] [Google Scholar]
  12. Ho-Terry L., Terry G. M., Londesborough P., Rees K. R., Wielaard F., Denissen A. Diagnosis of fetal rubella infection by nucleic acid hybridization. J Med Virol. 1988 Feb;24(2):175–182. doi: 10.1002/jmv.1890240206. [DOI] [PubMed] [Google Scholar]
  13. Hsia K., Spector D. H., Lawrie J., Spector S. A. Enzymatic amplification of human cytomegalovirus sequences by polymerase chain reaction. J Clin Microbiol. 1989 Aug;27(8):1802–1809. doi: 10.1128/jcm.27.8.1802-1809.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Kaneko S., Feinstone S. M., Miller R. H. Rapid and sensitive method for the detection of serum hepatitis B virus DNA using the polymerase chain reaction technique. J Clin Microbiol. 1989 Sep;27(9):1930–1933. doi: 10.1128/jcm.27.9.1930-1933.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Li H., Cui X., Arnheim N. Direct electrophoretic detection of the allelic state of single DNA molecules in human sperm by using the polymerase chain reaction. Proc Natl Acad Sci U S A. 1990 Jun;87(12):4580–4584. doi: 10.1073/pnas.87.12.4580. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. McClure M. A., Perrault J. Poliovirus genome RNA hybridizes specifically to higher eukaryotic rRNAs. Nucleic Acids Res. 1985 Oct 11;13(19):6797–6816. doi: 10.1093/nar/13.19.6797. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. McClure M. A., Perrault J. RNA virus genomes hybridize to cellular rRNAs and to each other. J Virol. 1986 Mar;57(3):917–921. doi: 10.1128/jvi.57.3.917-921.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Mullis K. B., Faloona F. A. Specific synthesis of DNA in vitro via a polymerase-catalyzed chain reaction. Methods Enzymol. 1987;155:335–350. doi: 10.1016/0076-6879(87)55023-6. [DOI] [PubMed] [Google Scholar]
  20. Nakhasi H. L., Meyer B. C., Liu T. Y. Rubella virus cDNA. Sequence and expression of E1 envelope protein. J Biol Chem. 1986 Dec 15;261(35):16616–16621. [PubMed] [Google Scholar]
  21. Okayama H., Berg P. High-efficiency cloning of full-length cDNA. Mol Cell Biol. 1982 Feb;2(2):161–170. doi: 10.1128/mcb.2.2.161. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Oldstone M. B. Viral persistence. Cell. 1989 Feb 24;56(4):517–520. doi: 10.1016/0092-8674(89)90573-4. [DOI] [PubMed] [Google Scholar]
  23. 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]
  24. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Simmonds P., Balfe P., Peutherer J. F., Ludlam C. A., Bishop J. O., Brown A. J. Human immunodeficiency virus-infected individuals contain provirus in small numbers of peripheral mononuclear cells and at low copy numbers. J Virol. 1990 Feb;64(2):864–872. doi: 10.1128/jvi.64.2.864-872.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
  27. Tingle A. J., Chantler J. K., Pot K. H., Paty D. W., Ford D. K. Postpartum rubella immunization: association with development of prolonged arthritis, neurological sequelae, and chronic rubella viremia. J Infect Dis. 1985 Sep;152(3):606–612. doi: 10.1093/infdis/152.3.606. [DOI] [PubMed] [Google Scholar]
  28. Tingle A. J. One infectious agent--many syndromes. J Rheumatol. 1987 Aug;14(4):653–655. [PubMed] [Google Scholar]
  29. Vidgren G., Takkinen K., Kalkkinen N., Käriäinen L., Pettersson R. F. Nucleotide sequence of the genes coding for the membrane glycoproteins E1 and E2 of rubella virus. J Gen Virol. 1987 Sep;68(Pt 9):2347–2357. doi: 10.1099/0022-1317-68-9-2347. [DOI] [PubMed] [Google Scholar]

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

RESOURCES