Development and validation of a Q-PCR based TCID50 method for human herpesvirus 6
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
Virology Journal 2012, 9:311 doi:10.1186/1743-422X-9-311Published: 18 December 2012
For titer assessment of human herpesvirus 6 (HHV-6), IFA targeting viral proteins or a TCID50 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.
We have developed and validated an alternative TCID50 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 TCID50 compared to 45% for ocular inspection read-out of TCID50, 14% for IFA read-out of TCID50, and 43% for an infectious units approach using IFA. The average inter-assay CV for quantitative PCR read-out of TCID50 was 73%, compared to 66%, 25% and 77% for the ocular inspection read-out for TCID50, IFA read-out of TCID50 and infectious unit approaches respectively.
The quantitative PCR based read-out of TCID50 proved to be more robust and easier to interpret than traditional TCID50 assessment approaches for HHV-6, and therefore it might be considered as an alternative method.