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. 1989 Aug;27(8):1802–1809. doi: 10.1128/jcm.27.8.1802-1809.1989

Enzymatic amplification of human cytomegalovirus sequences by polymerase chain reaction.

K Hsia 1, D H Spector 1, J Lawrie 1, S A Spector 1
PMCID: PMC267675  PMID: 2549097

Abstract

Polymerase chain reaction (PCR) amplification was used to detect human cytomegalovirus (HCMV) sequences. The fragments selected for amplification were fragments of 130 and 152 base pairs (bp) located at two opposite ends of HCMV strain AD169 EcoRI fragment D. Amplification of the 152-bp DNA was consistently greater than that of the 130-bp DNA. At the optimal Mg2+ concentration of 5 mM, specific PCR amplification of 152-bp DNA with Taq polymerase was sensitive; only one AD169-infected fibroblast cell or 0.01 pg of AD169 fragment D DNA was needed for detection. This specific amplification was also found with various clinical HCMV isolates and peripheral blood cells and urine from patients. In 37 urine samples analyzed simultaneously by PCR and by virus cultivation, identical results were found in 35 samples, while 2 scored positive only by PCR. This suggests that specific amplification of 152-bp DNA is sensitive and can be used for rapid detection of HCMV infections.

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Selected References

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

  1. Hirt B. Selective extraction of polyoma DNA from infected mouse cell cultures. J Mol Biol. 1967 Jun 14;26(2):365–369. doi: 10.1016/0022-2836(67)90307-5. [DOI] [PubMed] [Google Scholar]
  2. Kogan S. C., Doherty M., Gitschier J. An improved method for prenatal diagnosis of genetic diseases by analysis of amplified DNA sequences. Application to hemophilia A. N Engl J Med. 1987 Oct 15;317(16):985–990. doi: 10.1056/NEJM198710153171603. [DOI] [PubMed] [Google Scholar]
  3. 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]
  4. McMahon G., Davis E., Wogan G. N. Characterization of c-Ki-ras oncogene alleles by direct sequencing of enzymatically amplified DNA from carcinogen-induced tumors. Proc Natl Acad Sci U S A. 1987 Jul;84(14):4974–4978. doi: 10.1073/pnas.84.14.4974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. 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]
  6. Oste C. Polymerase chain reaction. Biotechniques. 1988 Feb;6(2):162–167. [PubMed] [Google Scholar]
  7. Ou C. Y., Kwok S., Mitchell S. W., Mack D. H., Sninsky J. J., Krebs J. W., Feorino P., Warfield D., Schochetman G. DNA amplification for direct detection of HIV-1 in DNA of peripheral blood mononuclear cells. Science. 1988 Jan 15;239(4837):295–297. doi: 10.1126/science.3336784. [DOI] [PubMed] [Google Scholar]
  8. Saiki R. K., Bugawan T. L., Horn G. T., Mullis K. B., Erlich H. A. Analysis of enzymatically amplified beta-globin and HLA-DQ alpha DNA with allele-specific oligonucleotide probes. Nature. 1986 Nov 13;324(6093):163–166. doi: 10.1038/324163a0. [DOI] [PubMed] [Google Scholar]
  9. 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]
  10. Saiki R. K., Scharf S., Faloona F., Mullis K. B., Horn G. T., Erlich H. A., Arnheim N. Enzymatic amplification of beta-globin genomic sequences and restriction site analysis for diagnosis of sickle cell anemia. Science. 1985 Dec 20;230(4732):1350–1354. doi: 10.1126/science.2999980. [DOI] [PubMed] [Google Scholar]
  11. Scharf S. J., Horn G. T., Erlich H. A. Direct cloning and sequence analysis of enzymatically amplified genomic sequences. Science. 1986 Sep 5;233(4768):1076–1078. doi: 10.1126/science.3461561. [DOI] [PubMed] [Google Scholar]
  12. Shibata D. K., Arnheim N., Martin W. J. Detection of human papilloma virus in paraffin-embedded tissue using the polymerase chain reaction. J Exp Med. 1988 Jan 1;167(1):225–230. doi: 10.1084/jem.167.1.225. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. 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]
  14. Spector D. H., Hock L., Tamashiro J. C. Cleavage maps for human cytomegalovirus DNA strain AD169 for restriction endonucleases EcoRI, BglII, and HindIII. J Virol. 1982 May;42(2):558–582. doi: 10.1128/jvi.42.2.558-582.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Spector S. A., Schmidt K., Ticknor W., Grossman M. Cytomegaloviruria in older infants in intensive care nurseries. J Pediatr. 1979 Sep;95(3):444–446. doi: 10.1016/s0022-3476(79)80532-6. [DOI] [PubMed] [Google Scholar]
  16. Wong C., Dowling C. E., Saiki R. K., Higuchi R. G., Erlich H. A., Kazazian H. H., Jr Characterization of beta-thalassaemia mutations using direct genomic sequencing of amplified single copy DNA. 1987 Nov 26-Dec 2Nature. 330(6146):384–386. doi: 10.1038/330384a0. [DOI] [PubMed] [Google Scholar]
  17. Wrischnik L. A., Higuchi R. G., Stoneking M., Erlich H. A., Arnheim N., Wilson A. C. Length mutations in human mitochondrial DNA: direct sequencing of enzymatically amplified DNA. Nucleic Acids Res. 1987 Jan 26;15(2):529–542. doi: 10.1093/nar/15.2.529. [DOI] [PMC free article] [PubMed] [Google Scholar]

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