Abstract
Background: Cytomegalovirus (CMV) causes life-threatening infections in immunocompromised patients, especially those with acquired immunodeficiency or organ transplants. Therefore, early detection of CMV is important to guide the clinical management of actively infected patients. Because detection of replicative transcripts indicates that the virus is in the process of being replicated in the infected cell, we applied a novel, sensitive, ligation-dependent (LD)-PCR method to detect CMV immediate-early (IE) messenger RNA (mRNA), an indicator of viral replication.
Methods and Results: Viral mRNAs were released from infected cells by incubation in 5 M guanidinium thiocyanate, and IE mRNAs were captured onto magnetic beads through oligo(dT) capture probes. Two hemiprobes, each containing an IE mRNA—complementary region and a region for PCR primer binding, were captured by binding to the IE mRNA. These hemiprobes, bound on an IE mRNA in juxtaposition to one another, were linked together by a DNA ligase to form a full probe that served as the template for PCR amplification. This approach detected IE mRNAs in CMV-propagating cells, but not in supernatants containing only viral DNAs. Thirty-one clinical specimens were tested by LD-PCR; 18 specimens were positive (ten specimens, bronchoalveolar lavage [BAL]; five specimens, urine; two specimens, blood; one specimen, biopsy), 17 of which were confirmed by culture. Three culture-positive samples (two specimens, urine; one specimen, BAL) were missed by LD-PCR, and one urine sample was positive by LD-PCR but negative by culture.
Conclusion: LD-PCR assay is a reliable test for the early diagnosis of active CMV infection in patient specimens.
Similar content being viewed by others
References
Patel R, Syndman DR, Rubin RH, et al.: Cytomegalovirus prophylaxis in solid organ transplant recipient. Transplantation 1996;61:1279–1289
Shibata D, Martin WJ, Appleman MD, Causey DM, Leedom JM, Arnheim N: Detection of cytomegalovirus DNA in peripheral blood of patients infected with human immunodeficiency virus. J Infect Dis 1988;158:1185–1192
Boeckh M, Bowden RA, Goodrich JM, Pettinger M, Meyers JD: Cytomegalovirus antigen detection in peripheral blood leukocytes after allogenic marrow transplantation. Blood 1992;80:1358–1364
Stenberg RM, Witte PR, Stinski MF: Multiple spliced and unspliced transcripts from human cytomegalovirus immediate-early region 2 and evidence for a common initiation site within immediate-early region 1. J Virol 1985;56:665–675
Randhawa PS, Manez R, Frye B, Ehrlich GD: Circulating immediate-early mRNA in patients with cytomegalovirus infections after solid organ transplantation. J Infect Dis 1994;170:1264–1267
Blok MJ, Goossens VJ, Vanherle SJV, et al.: Diagnostic value of monitoring human cytomegalovirus late pp67 mRNA expression in renal-allograft recipients by nucleic acid sequence-based amplification. J Clin Microbiol 1998;36:1341–1346
Gerna G, Baldanti F, Lilleri D, et al.: Human cytomegalovirus immediate-early mRNA detection by nucleic acid sequence-based amplification as a new parameter for preemptive therapy in bone marrow transplant recipients. J Clin Microbiol 2000;38:1845–1853
Hsuih TCH, Park YN, Zaretsky C, et al.: Novel, ligation-dependent PCR assay for detection of hepatitis C virus in serum. J Clin Microbiol 1996;34:501–507
Landry ML, Ferguson D: Comparison of quantitative cytomegalovirus antigenemia assay with culture methods and correlation with clinical disease. J Clin Microbiol 1993;31:2851–2856
Park YN, Abe K, Li H, Hsuih T, Thung SN, Zhang DY: Detection of hepatitis C virus RNA using ligation-dependent polymerase chain reaction in formalin-fixed, paraffin-embedded liver tissues. Am J Pathol 1996;149:1485–1491
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Wu, F., Li, H. & Zhang, D.Y. Differential Detection of Cytomegalovirus Immediate-early Messenger RNA in Clinical Samples Using Ligation-dependent PCR. Molecular Diagnosis 6, 233–239 (2001). https://doi.org/10.1007/BF03262059
Received:
Revised:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/BF03262059