title: Development and validation of a Q-PCR based TCID50 method for human herpesvirus 6.
abstract: BACKGROUND
  For titer assessment of human herpesvirus 6 (HHV-6), IFA targeting viral proteins or a TCID(50) method with ocular inspection for CPE can be used. These methods rely on the subjective decision of the assessor, obstructing the ability to obtain unanimous results.
  
  
  RESULTS
  We have developed and validated an alternative TCID(50) read-out approach where infection in the titration culture plate is assessed by viral DNA load change by quantitative PCR. A ten time increase in viral DNA load was determined as cut point for infection since that yielded a maximum correlation with viral protein expression (93%). The average intra-assay CV was 9% for quantitative PCR read-out of TCID(50) compared to 45% for ocular inspection read-out of TCID(50) , 14% for IFA read-out of TCID(50), and 43% for an infectious units approach using IFA. The average inter-assay CV for quantitative PCR read-out of TCID(50) was 73%, compared to 66%, 25% and 77% for the ocular inspection read-out for TCID(50), IFA read-out of TCID(50)and infectious unit approaches respectively.
  
  
  CONCLUSIONS
  The quantitative PCR based read-out of TCID(50)proved to be more robust and easier to interpret than traditional TCID(50)assessment approaches for HHV-6, and therefore it might be considered as an alternative method.
authors.affiliation: Karolinska Institutet, Department of Clinical Neuroscience, The Multiple Sclerosis Research Group, Center for Molecular Medicine building L8:00, Karolinska University hospital Solna, SE-171 76, Stockholm, Sweden. Rasmus.Gustafsson@ki.se
authors.forename: Rasmus K L
authors.initials: RK
authors.lastname: Gustafsson
authors.forename: Elin E
authors.initials: EE
authors.lastname: Engdahl
authors.forename: Anna
authors.initials: A
authors.lastname: Fogdell-Hahn
mesh_terms: D002460:Cell Line; D004279:DNA, Viral; D015654:Herpesvirus 6, Human / Q000235:genetics* / Q000254:growth & development; D006801:Humans; D060888:Real-Time Polymerase Chain Reaction; D015203:Reproducibility of Results; D019562:Viral Load / Q000379:methods*; D014779:Virus Replication
chemical_list: D004279:DNA, Viral