title: 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
  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.affiliation: Organon Teknika, Boxtel, Amsterdam, The Netherlands.
authors.forename: Astrid E
authors.initials: AE
authors.lastname: Greijer
authors.forename: Henriëtte M A
authors.initials: HM
authors.lastname: Adriaanse
authors.forename: Chantal A J
authors.initials: CA
authors.lastname: Dekkers
authors.forename: Jaap M
authors.initials: JM
authors.lastname: Middeldorp
mesh_terms: 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
chemical_list: 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