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

• Main Authors: Gustafsson Rasmus K L, Engdahl Elin E, Fogdell-Hahn Anna
• Format: Article
• Language: English
• Published: BMC 2012-12-01
• Series: Virology Journal
• Subjects: HHV-6A; Viral titer; TCID50; Q-PCR; Immunofluorescence assay; Ocular inspection
• Online Access: http://www.virologyj.com/content/9/1/311

<p>Abstract</p> <p>Background</p> <p>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.</p> <p>Findings</p> <p>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.</p> <p>Conclusions</p> <p>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.</p>