Multiplex real-time NASBA for monitoring expression dynamics of human cytomegalovirus encoded IE1 and pp67 RNA

• Published in: Journal of clinical virology 2002-02, Vol.24 (1), p.57-66
• Main Authors: Greijer, Astrid E., Adriaanse, Henriëtte M.A., Dekkers, Chantal A.J., Middeldorp, Jaap M.
• Format: Article
• Language: English
• Subjects: Antiviral intervention; Biological and medical sciences; Biomarkers - blood; Cytomegalovirus - isolation & purification; Cytomegalovirus - metabolism; Cytomegalovirus Infections - virology; Disease Progression; Fundamental and applied biological sciences. Psychology; Humans; Immediate-Early Proteins - genetics; Immediate-Early Proteins - metabolism; Infection monitoring; Lung Transplantation; Microbiology; Phosphoproteins - genetics; Phosphoproteins - metabolism; RNA, Messenger - blood; RNA, Viral - blood; Self-Sustained Sequence Replication; Traffic light; Viral Matrix Proteins - genetics; Viral Matrix Proteins - metabolism; Viral Proteins
• ISSN: 1386-6532; 1873-5967
• DOI: 10.1016/S1386-6532(01)00227-X

Background : The monitoring for HCMV mRNA expression in whole blood provides an accurate and informative diagnostic approach. Methods and materials: A multiplex real-time NASBA with three molecular beacons (MR-NASBA) was developed for the simultaneous detection and quantification of HCMV-encoded immediate early-1 (IE1) and late pp67 mRNA. The assay was evaluated using RNA from in vitro HCMV-infected cells and sequential whole blood samples (100 μl) of HCMV infected lung transplant recipients. Results: The MR-NASBA showed equal performance compared with standard NASBA assays (sensitivity of 1–3×10 3 RNA molecules in 100 μl blood and a linear range of 10 3–10 6 RNA molecules). The standard IE1 Q-RNA provides a reliable internal system control. No interference was observed between the individual beacon signals. The simultaneous ‘one-tube’ quantification of IE1-RNA levels combined with qualitative detection of pp67-RNA is feasible without loss of assay performance in clinical whole blood specimens. Conclusion and comments: MR-NASBA may be suitable for monitoring HCMV-activity in transplant recipients to aid in fine-tuning of antiviral intervention in high risk populations employing a traffic light diagnostic approach: no HCMV RNA signal (green light: safe) reflects absent or fully latent infection requiring no antiviral intervention; an ‘IE1-RNA only’ signal (yellow light: alert) indicates an emerging or subclinical active infection, opting for preemptive treatment in high risk populations; simultaneous ‘IE1-RNA plus pp67-RNA’ detection (red light:danger) indicates disseminating productive infection requiring immediate antiviral treatment.