Skip to main content
NCBI home page
Journal of Clinical Microbiology logoLink to Journal of Clinical Microbiology
. 1992 Mar;30(3):675–679. doi: 10.1128/jcm.30.3.675-679.1992

Early detection of bovine leukemia virus by using an enzyme-linked assay for polymerase chain reaction-amplified proviral DNA in experimentally infected cattle.

H M Naif 1, R C Daniel 1, W G Cougle 1, M F Lavin 1
PMCID: PMC265131  PMID: 1313047

Abstract

Bovine leukemia virus is the causative agent of bovine leukosis and has been described in many countries throughout the world. We describe here a sensitive and readily applicable assay for the detection of bovine leukemia proviral DNA. Detection relies on initial amplification of proviral DNA by using polymerase chain reaction (PCR) followed by an enzyme-linked assay (PCR-ELA). Amplification is carried out by using one biotinylated primer and a second primer containing the GCN4 protein binding site. DNA is detected by a colorimetric assay after it is coupled to GCN4-coated plates and subsequently incubated with horseradish-streptavidin peroxidase and the appropriate substrate to produce a chromogenic reaction. It was possible to detect proviral DNA for all of eight bovine leukemia virus-infected calves by 2 weeks postinfection. Use of the more conventional agar gel immunodiffusion assay failed to reveal the presence of the virus in any of the animals up to 4 weeks postinfection. The PCR-ELA detected as little as 0.1 to 0.2 ng of amplified DNA per well, which compares very favorably with ethidium bromide staining of gels, by which 1 to 2 ng per lane was detected. This method lends itself to mass screening, is carried out in a similar way to an enzyme-linked immunosorbent assay, and does not require gel electrophoresis or the use of radioactive gene probes.

Full text

PDF
675

Images in this article

677Image
on p.677
677Image
on p.677
678Image
on p.678

Selected References

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

  1. Brandon R. B., Naif H., Daniel R. C., Lavin M. F. Early detection of bovine leukosis virus DNA in infected sheep using the polymerase chain reaction. Res Vet Sci. 1991 Jan;50(1):89–94. doi: 10.1016/0034-5288(91)90059-w. [DOI] [PubMed] [Google Scholar]
  2. Coulston J., Naif H., Brandon R., Kumar S., Khan S., Daniel R. C., Lavin M. F. Molecular cloning and sequencing of an Australian isolate of proviral bovine leukaemia virus DNA: comparison with other isolates. J Gen Virol. 1990 Aug;71(Pt 8):1737–1746. doi: 10.1099/0022-1317-71-8-1737. [DOI] [PubMed] [Google Scholar]
  3. Eloit M., Kuzmak J., Dheiller M., Bénet J. J., Toma B. Comparison of performances of four ELISA kits in the detection of BLV antibodies in bulk tank milk or concentrated lactoserum from herds with low prevalence of infection. Biologicals. 1990 Jan;18(1):55–59. doi: 10.1016/1045-1056(90)90071-7. [DOI] [PubMed] [Google Scholar]
  4. Evermann J. F., DiGiacomo R. F., Ferrer J. F., Parish S. M. Transmission of bovine leukosis virus by blood inoculation. Am J Vet Res. 1986 Sep;47(9):1885–1887. [PubMed] [Google Scholar]
  5. Kemp D. J., Churchill M. J., Smith D. B., Biggs B. A., Foote S. J., Peterson M. G., Samaras N., Deacon N. J., Doherty R. Simplified colorimetric analysis of polymerase chain reactions: detection of HIV sequences in AIDS patients. Gene. 1990 Oct 15;94(2):223–228. doi: 10.1016/0378-1119(90)90391-4. [DOI] [PubMed] [Google Scholar]
  6. Kemp D. J., Smith D. B., Foote S. J., Samaras N., Peterson M. G. Colorimetric detection of specific DNA segments amplified by polymerase chain reactions. Proc Natl Acad Sci U S A. 1989 Apr;86(7):2423–2427. doi: 10.1073/pnas.86.7.2423. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Kwok S., Ehrlich G., Poiesz B., Kalish R., Sninsky J. J. Enzymatic amplification of HTLV-I viral sequences from peripheral blood mononuclear cells and infected tissues. Blood. 1988 Oct;72(4):1117–1123. [PubMed] [Google Scholar]
  8. Kwok S., Mack D. H., Mullis K. B., Poiesz B., Ehrlich G., Blair D., Friedman-Kien A., Sninsky J. J. Identification of human immunodeficiency virus sequences by using in vitro enzymatic amplification and oligomer cleavage detection. J Virol. 1987 May;61(5):1690–1694. doi: 10.1128/jvi.61.5.1690-1694.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Miller S. A., Dykes D. D., Polesky H. F. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 1988 Feb 11;16(3):1215–1215. doi: 10.1093/nar/16.3.1215. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. 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]
  11. Naif H. M., Brandon R. B., Daniel R. C., Lavin M. F. Bovine leukaemia proviral DNA detection in cattle using the polymerase chain reaction. Vet Microbiol. 1990 Nov;25(2-3):117–129. doi: 10.1016/0378-1135(90)90071-3. [DOI] [PubMed] [Google Scholar]
  12. Portetelle D., Bruck C., Mammerickx M., Burny A. Use of monoclonal antibody in an ELISA test for the detection of antibodies to bovine leukaemia virus. J Virol Methods. 1983 Jan;6(1):19–29. doi: 10.1016/0166-0934(83)90064-2. [DOI] [PubMed] [Google Scholar]
  13. Portetelle D., Mammerickx M., Burny A. Use of two monoclonal antibodies in an ELISA test for the detection of antibodies to bovine leukaemia virus envelope protein gp51. J Virol Methods. 1989 Feb;23(2):211–222. doi: 10.1016/0166-0934(89)90135-3. [DOI] [PubMed] [Google Scholar]
  14. Roberts D. H., Lucas M. H., Swallow C. Comparison of the agar gel immunodiffusion test and ELISA in the detection of bovine leukosis virus antibody in cattle persistently infected with bovine virus diarrhoea virus. Vet Immunol Immunopathol. 1989 Oct;22(3):275–281. doi: 10.1016/0165-2427(89)90014-7. [DOI] [PubMed] [Google Scholar]
  15. Rogers R. J., Dimmock C. K., de Vos A. J., Rodwell B. J. Bovine leucosis virus contamination of a vaccine produced in vivo against bovine babesiosis and anaplasmosis. Aust Vet J. 1988 Sep;65(9):285–287. doi: 10.1111/j.1751-0813.1988.tb16144.x. [DOI] [PubMed] [Google Scholar]
  16. Sagata N., Yasunaga T., Tsuzuku-Kawamura J., Ohishi K., Ogawa Y., Ikawa Y. Complete nucleotide sequence of the genome of bovine leukemia virus: its evolutionary relationship to other retroviruses. Proc Natl Acad Sci U S A. 1985 Feb;82(3):677–681. doi: 10.1073/pnas.82.3.677. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. 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]
  18. Shibata D., Martin W. J., Appleman M. D., Causey D. M., Leedom J. M., Arnheim N. Detection of cytomegalovirus DNA in peripheral blood of patients infected with human immunodeficiency virus. J Infect Dis. 1988 Dec;158(6):1185–1192. doi: 10.1093/infdis/158.6.1185. [DOI] [PubMed] [Google Scholar]
  19. Wu M. C., Shanks R. D., Lewin H. A. Milk and fat production in dairy cattle influenced by advanced subclinical bovine leukemia virus infection. Proc Natl Acad Sci U S A. 1989 Feb;86(3):993–996. doi: 10.1073/pnas.86.3.993. [DOI] [PMC free article] [PubMed] [Google Scholar]

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

RESOURCES