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.
Virol J. 2012 Dec 18;9:311. doi: 10.1186/1743-422X-9-311.
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.
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.
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.
对于人类疱疹病毒 6 (HHV-6) 的滴度评估，可以使用针对病毒蛋白的IFA 或具有 CPE 眼部检查的 TCID(50)方法。这些方法依赖于评估者的主观判断，阻碍了获得一致结果的能力。
我们已经开发并验证了一种替代的 TCID(50)读取方法，其中通过定量 PCR 评估滴定培养板中的感染是通过病毒 DNA 负载变化来评估的。病毒 DNA 负载增加十倍被确定为感染的临界点，因为这与病毒蛋白表达（93%）最大相关。与 TCID(50)的眼部检查读取相比，定量 PCR 读取 TCID(50)的平均内试验 CV 为 9%，而 TCID(50)的 IFA 读取为 45%，IFA 用于 TCID(50)的感染单位方法为 14%。定量 PCR 读取 TCID(50)的平均组间 CV 为 73%，而 TCID(50)的眼部检查读取、IFA 读取 TCID(50)和感染单位方法分别为 66%、25%和 77%。
与传统的 TCID(50)评估方法相比，基于定量 PCR 的 TCID(50)读取对于 HHV-6 更稳健且更易于解释，因此可以考虑作为替代方法。
