Laboratory of DNA Viruses, Division of Viral Products, OVRR, CBER, FDA, Bethesda, MD 20892, USA.
Virol J. 2013 Jun 15;10:195. doi: 10.1186/1743-422X-10-195.
Few studies have used quantitative polymerase chain reaction (qPCR) as an approach to measure virus neutralization assay endpoints. Its lack of use may not be surprising considering that sample nucleic acid extraction and purification can be expensive, labor-intensive, and rate-limiting.
Virus/antibody mixtures were incubated for one hour at 37°C and then transferred to Vero cell monolayers in a 96-well plate format. At 24 (or 48) hours post-infection, we used a commercially available reagent to prepare cell lysates amenable to direct analysis by one-step SYBR Green quantitative reverse transcription PCR using primers specific for the RSV-N gene, thereby obviating the need for cumbersome RNA extraction and purification. The neutralization titer was defined as the reciprocal of the highest dilution needed to inhibit the PCR signal by 90% when compared with the mean value observed in virus control wells in the absence of neutralizing antibodies.
We have developed a qPCR-based neutralization assay for human respiratory syncytial virus. Due to the sensitivity of qPCR in detecting virus replication, endpoints may be assessed as early as 24 hours post-infection. In addition, the dynamic range of qPCR provides a basis for the assay to be relatively robust to perturbations in input virus dose (i.e., the assay is in compliance with the Percentage Law).
This qPCR-based neutralization assay is suitable for automated high-throughput applications. In addition, our experimental approach may be generalizable for the rapid development of neutralization assays for other virus families.
很少有研究使用定量聚合酶链反应（qPCR）来测量病毒中和测定终点。考虑到样本核酸提取和纯化可能既昂贵又费力，而且是速度的限制因素，因此它未被广泛应用可能并不奇怪。
病毒/抗体混合物在 37°C 下孵育一小时，然后转移到 96 孔板格式的 Vero 细胞单层中。在感染后 24（或 48）小时，我们使用市售试剂制备细胞裂解物，这些裂解物适合通过一步 SYBR Green 定量逆转录 PCR 直接分析，从而避免了繁琐的 RNA 提取和纯化。中和滴度定义为与无中和抗体的病毒对照孔中观察到的平均值相比，抑制 PCR 信号降低 90%所需的最高稀释度的倒数。
我们已经开发了一种基于 qPCR 的人类呼吸道合胞病毒中和测定法。由于 qPCR 在检测病毒复制方面的灵敏度，终点可以在感染后 24 小时内进行评估。此外，qPCR 的动态范围为该测定提供了相对稳健的基础，可抵抗输入病毒剂量的波动（即，该测定符合百分比定律）。
这种基于 qPCR 的中和测定法适用于自动化高通量应用。此外，我们的实验方法可能适用于其他病毒家族的中和测定法的快速开发。
