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Retroviral vectors have become an indispensable tool in the modern molecular biology laboratory. They allow stable expression of a gene of interest in dividing cells and stable gene knock-down by expression of short hairpin RNA (shRNA). However, for gene therapy products and vaccines produced in continuous cell lines where downstream processing is minimal, it is crucial that the products and cells do not contain replication-competent retroviruses.
Currently, multiple methods for retroviral titer quantification are available. The quantification of the reverse transcriptase (RT) activity is a retroviral titration approach, which is associated with all retroviral particles. In these assays, add an exogenous RNA template to the viral supernatant and estimate RT activity by determining the amount of RNA that is converted to cDNA by the retroviral RT. In the first generation RT assays, cDNA production was monitored through measuring labeled nucleotide incorporation. Sensitivity was greatly increased when a PCR amplification step of the synthesized cDNA was introduced prior to product detection. These types of assays are known as product-enhanced RT (PERT) assays. The newest PERT generation uses integrated qPCR techniques for fast cDNA quantification, further increasing the accuracy and linear range of the assays. Most qPCR based PERT assays use cDNA-specific fluorogenic labeled probes for signal generation (F-PERT). Recently, a one-step PERT assay using the more accessible and cost-efficient SYBR Green-I chemistry (SG-PERT) was also developed. PERT assays are now usually used for detection of retroviral contaminants in biological products intended for human use.
Figure 1. The principle of the SG-PERT assay.
(1) Production of replication-competent virus
Replication-competent virus was produced by transfection of cells with the proviral constructs. Transfection was performed with Transfection Kit, according to manufacturer’s instructions. Viral supernatant was harvested 48 hours or 72 hours after transfection and centrifugated at 900 g for 10 min, to clarify the supernatant from remaining cells. The high-titer viral supernatant, which was used to produce a standard curve for the SG-PERT assay, was obtained by infection of cells with virus and subsequent collection of the culture medium 12 days after infection. During infection, culture medium was refreshed every two or three days.
(2) SG-PERT assay
Cell-free viral supernatant was generally used without prior dilution as input for the assay. 10-fold dilution series of viral supernatant or virus recombinant RT were generated in IMDM complete. 5 μL of the viral supernatant or recombinant RT solution was added to a well of a 96-well U-bottom plate and mixed with 5 μL of 2× concentrated lysis buffer, already containing RNase inhibitor. Samples were incubated for 10 minutes at room temperature and then diluted by addition of 90 μL nuclease-free water. After centrifugation, the lysates were resuspended and used as input for the assay.
All reagents were kept on ice during preparation of the assay. For every sample lysate, an SG-PERT reaction was always performed in duplo. Cycles of quantification (Cq) values were generated through the software of the qPCR instruments.
In order to perform absolute quantification of RT activity values, a standard curve of replication-competent virus containing supernatant with known RT activity levels was run in parallel in each assay and values were extrapolated from the obtained Cq values. The standard curve was produced through serial dilution of a large batch of high-titer supernatant. Dilutions were aliquoted for using in different SG-PERT assays, to avoid loss of RT activity through repeated freeze-thaw cycles. RT activity values of the standard curve were determined by running a dilution series of commercial recombinant RT in parallel in at least four independent experiments.
(3) Calculations and statistics
Standard deviation (STDEV), the coefficient of determination (R2) and regression equations were calculated. Coefficient of variation (CV) was worked out as follows: (STDEV/AVERAGE) x 100%. Statistical significance of difference between inter-run coefficient of variation of ELISA test and SG-PERT assay was analyzed with the one-tailed nonparametric Mann-Whitney U test.