Multiplex real-time NASBA for monitoring expression dynamics of human cytomegalovirus encoded IE1 and pp67 RNA.
BACKGROUND
The monitoring for HCMV mRNA expression in whole blood provides an accurate and informative diagnostic approach.


METHODS
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.
Greijer
Astrid E
AE
Organon Teknika, Boxtel, Amsterdam, The Netherlands.
Adriaanse
Henriëtte M A
HM
Dekkers
Chantal A J
CA
Middeldorp
Jaap M
JM
D015415:Biomarkers / Q000097:blood; D003587:Cytomegalovirus / Q000302:isolation & purification / Q000378:metabolism*; D003586:Cytomegalovirus Infections / Q000821:virology*; D018450:Disease Progression; D006801:Humans; D017874:Immediate-Early Proteins / Q000235:genetics / Q000378:metabolism*; D016040:Lung Transplantation; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D012333:RNA, Messenger / Q000097:blood; D012367:RNA, Viral / Q000097:blood; D021161:Self-Sustained Sequence Replication; D014763:Viral Matrix Proteins / Q000235:genetics / Q000378:metabolism*; D014764:Viral Proteins
D015415:Biomarkers; C088645:IE1 protein, cytomegalovirus; D017874:Immediate-Early Proteins; D010750:Phosphoproteins; D012333:RNA, Messenger; D012367:RNA, Viral; D014763:Viral Matrix Proteins; D014764:Viral Proteins; C065418:cytomegalovirus matrix protein 65kDa