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. 2012 Dec 18:9:311.
doi: 10.1186/1743-422X-9-311.

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

Rasmus K L Gustafsson  1 , Elin E Engdahl, Anna Fogdell-Hahn
Affiliations

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

Rasmus K L Gustafsson et al. Virol J. .

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.

Findings: 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.

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Figures

Figure 1
Figure 1
Replication of HHV-6A (GS strain) followed by Q-PCR. Data shown are mean results (± SEM) of triplicate cultures for every multiplicity of infection (MOI). Connecting lines discontinue when the viral DNA load dropped under the detection limit.
Figure 2
Figure 2
Determination of cut-point for infection. The optimal cut-point for infection was found to be a ten times increase where the correlation to protein expression was seen in 93% of the wells by IFA with an antibody targeting the late viral protein gp116/54/64. No well contained viral protein where the viral DNA load had not increased ten times. Data shown are mean results (± SEM) from three TCID50 plates.
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