Abstract
We evaluated a rapid and sensitive method to determine human cytomegalovirus (CMV) DNA levels in blood cells using a quantitative polymerase chain reaction (PCR) technique. This method is based on real‐time detection of PCR using a dual fluorescence‐labeled probe and a sequence detector. Ten copies of CMV DNA were detected, when 1 μg of DNA from blood samples was used with this method, and a good correlation was obtained between increased concentrations of copy numbers calculated and measured copy numbers of CMV DNA (r = 0.999). Forty normal subjects exhibited no copies of CMV DNA. On the other hand, a 6‐month‐old girl tested positive for increased levels 4 weeks after liver transplant. This method is simple, accurate, and sensitive for the quantitative detection of CMV DNA in vivo, indicating possible applications for the diagnosis and monitoring of CMV infection. J. Clin. Lab. Anal. 15:122–126, 2001. © 2001 Wiley‐Liss, Inc.
Keywords: polymerase chain reaction (PCR), quantitative PCR, cytomegalovirus (CMV), liver transplantation
REFERENCES
- 1. Olive DM, Simsek M, AL‐mufti S. 1989. Polymerase chain reaction assay for detection of human cytomegalovirus. J Clin Microbiol 27:1238–1242. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2. Cassol SA, Poon M‐C, Pal R, et al. 1989. Primer‐mediated enzymatic amplification of cytomegalovirus (CMV) DNA. Application of the early diagnosis of CMV infection in marrow transplant recipients. J Clin Invest 83:1109–1115. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 3. Diviacco S, Norio P, Zentilin L, et al. 1992. A novel procedure for quantitative polymerase chain reaction by coamplication of competitive templates. Gene 122:313–320. [DOI] [PubMed] [Google Scholar]
- 4. Zipeto D, Baldanti F, Zella D, et al. 1993. Quantification of human cytomegalovirus DNA in peripheral blood polymorphonuclear leukocytes of immunocompromised patients by the polymerase chain reaction. J Virol Methods 44:45–56. [DOI] [PubMed] [Google Scholar]
- 5. Holland P, Abramson RD, Watson R, Gelfand DH. 1991. Detection specific exonuclease activity of Thermus aquatisus DNA polymerase. Proc Natl Acad Sci U S A 88:7276–7280. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6. Lyamichev V, Brow MAD, Dahlverg JE. 1993. Structure‐specific endonucleolytic cleavage of nucleic acids by eubacterial DNA polymerase. Science 260:778–783. [DOI] [PubMed] [Google Scholar]
- 7. Livak KJ, Flood SJA, Marmoro J, Giusti W, Deetz K. 1995. Oligonucleotides with fluorescent dyes at opposite ends provide a quenched probe system useful for detecting PCR product and nucleic acid hybridization. PCR Methods Appl 4:357–362. [DOI] [PubMed] [Google Scholar]
- 8. Pritchett RF. 1980. DNA nucleotide sequence heterogeneity between the Towne and AD169 strains of cytomegalovirus. J Virol 36:152–161. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 9. Maniatis T, Fritsch EF, Sambrook J. Molecular cloning: a laboratory manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory; 1982; p 85–93. [Google Scholar]
- 10. Gilliland G, Perrin S, Blanchard K, Bunn HF. 1990. Analysis of cytokine mRNA and DNA: detection and quantitation by competitive polymerase chain reaction. Proc Natl Acad Sci U S A 87:2725–2729. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11. Siebert PD, Larrick JW. 1993. PCR MINICS: competitive DNA fragment for use as internal standards in quantitative PCR. BioTechniques 14:244–249. [PubMed] [Google Scholar]
- 12. Lear W, Mcdonnell M, Kashyap S, Boer PH. 1995. Random primer p(dN)6‐digoxigenin labeling for quantitation of mRNA by Q‐RT‐PCR and ELISA. BioTechniques 18:78–83. [PubMed] [Google Scholar]