Development and validation of a Q-PCR based TCID50 method for human herpesvirus 6.
| Autor: | Gustafsson RK; 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, Engdahl EE, Fogdell-Hahn A |
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| Jazyk: | angličtina |
| Zdroj: | Virology journal [Virol J] 2012 Dec 18; Vol. 9, pp. 311. Date of Electronic Publication: 2012 Dec 18. |
| DOI: | 10.1186/1743-422X-9-311 |
| Abstrakt: | 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. |
| Databáze: | MEDLINE |
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