Development and validation of a Q-PCR based TCID50 method for human herpesvirus 6

作者：Gustafsson Rasmus K L^*; Engdahl Elin E; Fogdell Hahn Anna

来源：Virology Journal, 2012, 9(1): 311.

DOI：10.1186/1743-422X-9-311

• HHV-6A
• Viral titer
• TCID50
• Q PCR
• Immunofluorescence assay
• Ocular inspection

Background: 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.
Findings: 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.
Conclusions: 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.

• 出版日期2012-12-18