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. 2002 Nov 12;29(3):243–255. doi: 10.1016/0166-0934(90)90052-H

Polymerase chain reaction for probe synthesis and for direct amplification in detection of bovine coronavirus

Arnold Verbeek 1, Peter Tijssen 1,∗
PMCID: PMC7119836  PMID: 2266145

Abstract

The polymerase chain reaction (PCR) was used to synthesize ds and ss probes for the detection of bovine coronavirus (BCV) using recombinant plasmids as template molecules. The ds probes detected a minimum of about 2 × 105 viral genomes after exposure for 1 h, a detection limit similar to nick-translated probes after exposure of the films for 60 h. More than 8 h exposure to blots probed with these ds probes resulted in complete darkening of the film. The ss probes, synthesized by asymmetric PCR on linearized plasmids, permitted the detection of 5 × 104 genomes, which equalled the capacity of random-primed probes. Prolonged exposure did not increase the background as in case of ds PCR-probed blots. Probes, synthesized by asymmetric PCR and random-priming were labeled to similar specific activities and were better in terms of sensitivity and detectability as opposed to nick-translated probes. However, the specificity of detection with ss probes as to random primed probes was increased further. About 10 viral genomes, after fragment-specific amplification by PCR, were detected by agarose-gel analysis. PCR-probe synthesis was simple, highly reproducible, and allowed the synthesis of probes for specific fragments.

Keywords: Bovine coronavirus, Polymerase chain reaction, Probe labeling, Detection

References

  1. Albretsen C., Haukanus B., Aasland R., Kleppe K. Optimal conditions for hybridization with oligonucleotides: a study with myc-oncogene DNA probes. Anal. Biochem. 1988;170:193–202. doi: 10.1016/0003-2697(88)90108-x. [DOI] [PubMed] [Google Scholar]
  2. Ambinder R.F., Charache P., Staal S., Wright P., Forman M., Hayward D.S., Hayward G.S. The vector homology problem in diagnostic nucleic acid hybridization of clinical specimens. J. Clin. Microbiol. 1986;24:16–20. doi: 10.1128/jcm.24.1.16-20.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Barnett B.B., Splendlove R.S., Peterson M.W., Hsu L.Y., LaSalle V.A., Egbert L.N. Immunofluorescent cell assay of neonatal calf diarrhea virus. Can. J. Comp. Med. 1975;39:462–465. [PMC free article] [PubMed] [Google Scholar]
  4. Bradford M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem. 1976;72:248–254. doi: 10.1006/abio.1976.9999. [DOI] [PubMed] [Google Scholar]
  5. Bridger J.C., Woode G.N., Meyling A. Isolation of coronaviruses from neonatal calf diarrhea in Great Britain and Denmark. Vet. Microbiol. 1978;3:101–114. [Google Scholar]
  6. Bruce C.B., Al-Nahib W., Almond J.W., Tyrrell D.A. Use of synthetic oligonucleotide probes to detect rhinovirus RNA. Arch. Virol. 1989;105:179–187. doi: 10.1007/BF01311355. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Collins M.L., Hunsaker W.R. Improved hybridization assays employing tailed oligonucleotide probes: A direct comparison with 5' end-labeled oligonucleotide probes and nick-translated plasmid probes. Anal. Biochem. 1985;151:211–224. doi: 10.1016/0003-2697(85)90168-x. [DOI] [PubMed] [Google Scholar]
  8. Crouch C.F., Raybould T.J.G., Acres S.D. Monoclonal antibody capture enzyme-linked immunosorbent assay for detection of enteric bovine coronavirus. J. Clin. Microbiol. 1984;19:388–393. doi: 10.1128/jcm.19.3.388-393.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Dea S., Roy R.S., Begin M.E. Physiochemical and biological properties of neonatal calf diarrhea coronaviruses isolated in Quebec and comparison with the Nebraska calf coronavirus. Am. J. Vet. Res. 1980;41:23–29. [PubMed] [Google Scholar]
  10. Diegutis P.S., Keirnan E., Burnett L., Nightingale B.N., Cossart Y.E. False-positive results with hepatitis B virus DNA dot-blot hybridization in hepatitis B surface antigen-negative specimens. J. Clin. Microbiol. 1986;23:779–794. doi: 10.1128/jcm.23.4.797-799.1986. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. El-Ghorr A.A., Snodgrass D.R., Scott F.M.M. Evaluation of an immunogold electron microscopy technique for detecting bovine coronavirus. J. Virol. Methods. 1988;19:215–224. doi: 10.1016/0166-0934(88)90016-X. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Feinberg A.P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 1983;132:6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
  13. Gyllensten U.B., Erlich H.A. Vol. 85. 1988. Generation of single-stranded DNA by the polymerase chain reaction and its application to direct sequencing of the HLA-DQA locus; pp. 7652–7656. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Laporte J., Bobulesco P., Rossi F. Une lignée cellulaire particulièrement sensible à la replication du coronavirus ent/'erique bovin: les cellules HRT-18. Comptes Rendus Acad. Sci. Paris. 1980;290:623–626. [PubMed] [Google Scholar]
  15. Lapps W., Hogue B.G., Brian D.A. Sequence analysis of the bovine coronavirus nucleocapsid and matrix genes. Virology. 1987;157:47–57. doi: 10.1016/0042-6822(87)90312-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Leary J.J., Brigati D.J., Ward D.C. Vol. 80. 1983. Rapid and sensitive colorimetric method for visualizing biotin-labeled probes hybridized to DNA or RNA immobilized on nitrocellulose: bioblots; pp. 4045–4049. (Proc. Natl. Acad. Sci. USA). [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Maniatis T., Fritsch E.F., Sambrook J. Cold Spring Harbor Laboratory; Cold Spring Harbor, New York: 1982. (Molecular Cloning: A Laboratory Manual). [Google Scholar]
  18. Mebus C.A., Stair E.L., Rhodes M.B., Twiehaus M.J. Pathology of neonatal calf diarrhea induced by a corona-like agent. Vet. Pathol. 1973;10:45–64. doi: 10.1177/030098587301000105. [DOI] [PubMed] [Google Scholar]
  19. Meinkoth J., Wahl G. Hybridization of nucleic acids immobilized on solid supports. Ann. Biochem. 1984;138:267–284. doi: 10.1016/0003-2697(84)90808-x. [DOI] [PubMed] [Google Scholar]
  20. Myint S., Siddell S., Tyrrell D. The use of nucleic acid hybridization to detect human coronaviruses. Arch. Virol. 1989;104:335–337. doi: 10.1007/BF01315554. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Reynolds D.J., Chasey D., Scott A.C., Bridges J.C. Evaluation of ELISA and electron microscopy for detection of coronavirus and rotavirus in bovine faeces. Vet. Rec. 1984;114:397–401. doi: 10.1136/vr.114.16.397. [DOI] [PubMed] [Google Scholar]
  22. Rigby P.W., Dieckman M., Rhodes C., Berg P. Labeling deoxyribonucleic acid to high specific activity in vitro by nick translation with DNA polymerase I. J. Mol. Biol. 1977;113:237–251. doi: 10.1016/0022-2836(77)90052-3. [DOI] [PubMed] [Google Scholar]
  23. Saif L., Redman D., Moorhead P., Theil K. Experimentally induced coronavirus infections in calves; viral replication in the respiratory and intestinal tracts. Am. J. Vet. Res. 1986;47:1426–1432. [PubMed] [Google Scholar]
  24. Saiki R.K., Gelfand D.H., Stoffel S., Scharf S.J., Highuchi R., Horn G.T., Mullis K.B., Erlich H.A. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 1988;239:487–491. doi: 10.1126/science.2448875. [DOI] [PubMed] [Google Scholar]
  25. Sato K., Inaba Y., Tokohisa S., Miura Y., Kaneko N., Asagi M., Matumo M. Detection of bovine coronavirus in feces by reverse passive hemagglutination. Arch. Virol. 1984;80:23–31. doi: 10.1007/BF01315291. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Schowalter D.B., Sommer S.S. The generation of radiolabeled DNA and RNA probes with polymerase chain reaction. Ann. Biochem. 1989;177:90–94. doi: 10.1016/0003-2697(89)90019-5. [DOI] [PubMed] [Google Scholar]
  27. Shockley L.J., Kapke P.A., Lapps W., Brian D.A., Potgieter L.N.D., Woods R. Diagnosis of porcine and bovine coronavirus infection using cloned cDNA probes. J. Clin. Microbiol. 1987;25:1591–1596. doi: 10.1128/jcm.25.9.1591-1596.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Van Balken J.M., de Leeuw P.W., Ellens D.J., Straver P.J. Detection of coronavirus in calf faeces with a haemadsorption-elution-haemagglutination assay (HEHA) Vet. Microbiol. 1978/1979;3:205–211. [Google Scholar]
  29. Van Dyke B.V., Murphy-Corb M. Detection of respiratory syncytial virus in nasopharyngeal secretion by DNA-RNA hybridization. J. Clin. Microbiol. 1989;27:1739–1743. doi: 10.1128/jcm.27.8.1739-1743.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Verbeek A., Tijssen P. Biotinylated and radioactive cDNA probes in the detection by hybridization of bovine enteric coronavirus. Mol. Cell. Probes. 1988;2:209–223. doi: 10.1016/0890-8508(88)90005-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Verbeek A., Dea S., Tijssen P. Detection of bovine enteric coronavirus in clinical specimens by hybridization with cDNA probes. Mol. Cell. Probes. 1990;4:107–120. doi: 10.1016/0890-8508(90)90012-O. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Zeff R.A., Geliebter J. Vol. 9. BRL Press; 1987. Oligonucleotide probes for genomic DNA blots; pp. 1–2. (Focus). No. 2. [Google Scholar]

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