Differential detection of cytomegalovirus immediate-early messenger RNA in clinical samples using ligation-dependent PCR

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[ndash ]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.