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

Abstract

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 microl) of HCMV infected lung transplant recipients.

RESULTS:

The MR-NASBA showed equal performance compared with standard NASBA assays (sensitivity of 1-3 x 10(3) RNA molecules in 100 microl 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.

• Authors: Astrid E Greijer, Henriëtte M A Adriaanse, Chantal A J Dekkers, Jaap M Middeldorp
• Journal: Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology (J Clin Virol) Vol. 24 Issue 1-2 Pg. 57-66 (Feb 2002) ISSN: 1386-6532 [Print] Netherlands
• PMID: 11744429 (Publication Type: Comparative Study, Journal Article)

Chemical References

• Biomarkers
• IE1 protein, cytomegalovirus
• Immediate-Early Proteins
• Phosphoproteins
• RNA, Messenger
• RNA, Viral
• Viral Matrix Proteins
• Viral Proteins
• cytomegalovirus matrix protein 65kDa

Topics

• Biomarkers (blood)
• Cytomegalovirus (isolation & purification, metabolism)
• Cytomegalovirus Infections (virology)
• Disease Progression
• Humans
• Immediate-Early Proteins (genetics, metabolism)
• Lung Transplantation
• Phosphoproteins (genetics, metabolism)
• RNA, Messenger (blood)
• RNA, Viral (blood)
• Self-Sustained Sequence Replication
• Viral Matrix Proteins (genetics, metabolism)
• Viral Proteins