Measuring the Infectious Titer of Recombinant Adenovirus Using Tissue Culture Infection Dose 50% (TCID50) End-Point Dilution and Quantitative Polymerase Chain Reaction (qPCR).
Measuring the Infectious Titer of Recombinant Adenovirus Using Tissue Culture Infection Dose 50% (TCID50) End-Point Dilution and Quantitative Polymerase Chain Reaction (qPCR).
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DOI：
10.1101/pdb.prot095562
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发表时间：
2019-08-01
影响因子：
--
通讯作者：
Gao, Guangping
中科院分区：
文献类型：
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作者：
Lock, Martin;Korn, Michael;Gao, Guangping
传统上，腺病毒和重组腺病毒感染滴度通过空斑测定来测量，其中用连续稀释的腺病毒原液感染细胞，然后用琼脂覆盖;空斑将作为单一感染事件的结果形成。尽管该方法给出了定量读数（针对稀释度校正的噬菌斑数目），但可能存在灵敏度和再现性的问题，特别是当使用感染标准细胞系的效率差的腺病毒血清型时。另一种方法是将在96孔组织培养板的威尔斯孔中生长的细胞的系列稀释液铺板，并确定50%的威尔斯孔被感染的稀释度。这种古老而可靠的技术被称为“组织培养感染剂量50%”（TCID 50）终点稀释法，已用于滴定许多病毒，特别是那些不容易形成噬斑的病毒。通常，感染的威尔斯孔通过直接检查细胞病变效应（CPE）或细胞活力来确定。然而，通过结合96孔TCID 50格式和定量聚合酶链反应（qPCR）的检测能力，可以实现灵敏度的大幅提高-在我们手中10倍，具有一系列转基因和腺病毒血清型。该方案使用96孔TCID 50格式，结合qPCR进行灵敏和定量阳性孔调用，以确定腺病毒载体的感染滴度。
Traditionally, adenovirus and recombinant adenovirus infectious titers have been measured by plaque assay, in which the cells are infected with serially diluted adenovirus stock and then overlaid with agar; a plaque will form as the result of a single infectious event. Although this method gives a quantitative readout (number of plaques corrected for the dilution), there can be issues with sensitivity and reproducibility, especially when adenovirus serotypes are used that infect standard cell lines with poor efficiency. An alternative approach is to plate serial dilutions of the cells growing in the wells of a 96-well tissue culture plate and determine the dilution at which 50% of the wells are infected. This ancient and reliable technique known as the "tissue culture infection dose 50%" (TCID50) end-point dilution method has been used for titering a number of viruses, especially those that do not readily form plaques. Usually, infected wells are determined by direct examination for cytopathic effect (CPE) or cell viability. However, by combining a 96-well TCID50 format and the power of quantitative polymerase chain reaction (qPCR) for detection, a large increase in sensitivity-in our hands 10-fold, with a range of both transgenes and adenovirus serotypes-can be achieved. This protocol uses a 96-well TCID50 format, in conjunction with qPCR for sensitive and quantitative positive-well calling, to determine infectious titer of adenovirus vectors.