Figure 1.
Principle of the SG-PERT assay.
Cell-free retrovirus containing supernatant is lysed and added to a reaction mix containing the MS2 RNA template, MS2 complementary primers and a SYBR Green I qPCR mastermix. During a one-step reaction, the reverse transcriptase (RT) enzymes derived from the retroviral particles will convert the MS2 RNA into cDNA and cDNA is subsequently quantified by qPCR amplification of the MS2 cDNA. The amount of synthesized cDNA represents the level of RT activity in the viral supernatant and is thereby a measure of the amount of retroviral particles.
Figure 2.
Sensitivity and specificity of the SG-PERT assay.
(A) Melting curves of PCR products obtained by SG-PERT assay on the LightCycler® 480 when using 1011 or 104 pU recombinant HIV-1 RT or nuclease-free water (non-template control = NTC) as input for the assay, as indicated. (B, D) Amplification curves of indicated amount of (B) recombinant HIV-1 RT (pU), (D) replication competent HIV-1 (NL4-3 strain) (ng p24/mL) or nuclease-free water (NTC) obtained by SG-PERT on the LightCycler® 480. (C,E) Relation between input of (C) recombinant HIV-1 RT, (E) replication competent HIV-1 (HIV-1), HIV-1 based lentiviral vectors (HIV-1 based vector) or Moloney Murine Leukemia-based retroviral vectors (MoMLV) and obtained cycle of quantification (Cq) values by SG-PERT on the LightCycler® 480. Viral titers in the undiluted samples in (E) (value of “0” on x-axis) were 3,100 ng p24/mL for the replication competent HIV-1 virus, 1.12×107 transducing units/mL (TU/mL) for the HIV-1 based viral vector and 4.9×105 TU/mL for the MoMLV-based vector. Only input levels within linear range of the assay were included for correlation analysis.
Figure 3.
Intra- and inter-run variation of the SG-PERT assay.
(A) Standard curve composed of a pre-made six 10-fold serial dilution series of replication-competent HIV-1 containing supernatant measured in 12 independent SG-PERT experiments. For each experiment obtained Cq values are plotted versus the RT activity in each sample. RT activity values were determined by running a dilution series of recombinant HIV-1 RT in parallel. Standard deviation on the obtained crossing point values is indicated for each dilution. (B) RT activity values obtained for 8 repeated measurements of different HIV-1 samples (sample number 1 to 6) within the same run. The average RT activity value for each sample is indicated by a red line, error bars represent standard deviation on the obtained RT activity values. Numbers indicate intra-run variation for each sample, expressed as percentage of the average RT activity values (coefficient of variation). (C) RT activity values obtained for different HIV-1 samples (sample number 1 to 11) in at least 3 independent SG-PERT experiments. The average RT activity value for each sample is indicated by a red line, error bars represent standard deviation on the obtained RT activity values. Numbers indicate inter-run variation for each sample, expressed as percentage of the average RT activity values (coefficient of variation). Experiments were performed on the LightCycler® 480.
Table 1.
Evaluation of different lentiviral titration methods.
Figure 4.
Comparison of SG-PERT assay with other lentiviral titration methods.
(A) Correlation between RT activity levels measured with SG-PERT and p24 antigen concentration levels measured with ELISA in supernatant containing replication-competent HIV-1 virus or HIV-1 based MISSION lentiviral vectors. Coefficient of determination and correlation functions are shown for HIV-1 virus (- - -) and lentiviral vectors (-·-·) separately or combined (-). To avoid introduction of interrun variation, p24 ELISA measurements for all samples were performed in the same experiment. (B) Correlation between RT activity levels measured by SG-PERT and levels of transducing units determined by limiting dilution on Jurkat E6.1 cells in HIV-1 based MISSION® lentiviral vector preparations. (C) Relation between the level of β-galactosidase activity, as a marker of HIV-1 Tat production and the relative amount (serial dilution) of replication competent HIV-1 NL4-3 virus used to infect the cells, in the culture of P4.R5 MAGI indicator cells 48 h after single-cycle infection. Viral input in the undiluted samples (value of “1” on x-axis) was 1.76×103 mU RT/mL for HIV sup 12 and 2.33×102 mU RT/mL for HIV sup 13. For HIV sup 12, the undiluted sample was outside the linear range of the assay and was therefore not included for calculation of the determination coefficient (R2) shown in panel C. (D) Correlation between the level of β-galactosidase activity in the culture of P4.R5 MAGI indicator cells 48 h after single-cycle infection with different productions of HIV-1 NL4-3 supernatant and the RT activity measured in the supernatant by SG-PERT. (E) p24 antigen concentration values obtained for different HIV-1 containing samples in at least 3 independent ELISA experiments. Sample numbers in x-axis correspond to those in Figure 3C. The average p24 concentration value for each sample is indicated by a red line, error bars represent standard deviation on the obtained p24 concentration values. Numbers indicate inter-run variation for each sample, expressed as percentage of the average p24 concentration (coefficient of variation = CV). (F) Average inter-run coefficient of variation for SG-PERT and p24 ELISA assays, calculated from the CV’s obtained in Figure 3C and Figure 4E respectively. Error bars indicate standard deviations. The asterix (*) indicates a statistical significant difference between the two values according to a one-tailed Mann-Withney U test (p-value = 0.0426).
Figure 5.
SG-PERT assay on ABI 7300 Real-Time PCR System.
(A) Amplification curves of indicated amount of replication competent HIV-1 (NL4-3 strain) (ng p24/mL) obtained by SG-PERT on the ABI 7300 instrument. The horizontal line represents the threshold line used to calculate Cq values. (B) Correlation between input levels of replication-competent HIV-1 virus and Cq values obtained by SG-PERT on the ABI 7300 instrument. p24 antigen concentration in the undiluted samples (value “0” on x-axis) was 3,100 ng/mL.