Multiplex real-time NASBA for monitoring expression dynamics of human cytomegalovirus encoded IE1 and pp67 RNA
| Autor: | Henriette Maria Aleida Adriaanse, Astrid E. Greijer, Chantal A. J. Dekkers, Jaap M. Middeldorp |
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| Rok vydání: | 2002 |
| Předmět: |
Human cytomegalovirus
viruses Cytomegalovirus Biology Immediate early protein Immediate-Early Proteins Viral Matrix Proteins Viral Proteins Molecular beacon Virology medicine Humans Multiplex RNA Messenger Self-Sustained Sequence Replication Whole blood Messenger RNA RNA Phosphoproteins medicine.disease NASBA Infectious Diseases Cytomegalovirus Infections Immunology Disease Progression RNA Viral Biomarkers Lung Transplantation |
| Zdroj: | Journal of Clinical Virology. 24:57-66 |
| ISSN: | 1386-6532 |
| DOI: | 10.1016/s1386-6532(01)00227-x |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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