Figure 1.
Threshold cycle (Ct) vs. log10(dilution factor) from SYBR Green qRT-PCR (targeting the M1 gene of influenza A viruses) applied to a serial dilution of total RNA purified from MDCK-London cells infected with A/PR/8/34 (n = 3 for each dilution), which was used as an RNA quantification standard for our experiments.
Lysate from uninfected MDCK-London cells prepared with the Bio-Rad iScript Sample Preparation Reagent (SPR) was used as the diluent to prepare the RNA serial dilution in order to achieve comparability with experimental samples. The initial dilution contained 10 ng of standard RNA per µL.
Figure 2.
Replication kinetics of influenza virus assessed by qRT-PCR.
Virus replication was assessed in the absence or presence of TPCK-trypsin (1 µg/mL). Virus (1000 TCID50 in 100 µL) was placed into a well of a 96-well plate. After incubation for 1 h at 37°C, a suspension of MDCK-London cells (30,000 in 100 µL) was added. At the indicated times, experimental samples were prepared using SPR and subjected to qRT-PCR. The RNA copy numbers were normalized to the mean value observed for A/PR/8/34 at 6 hours in the presence of TPCK-trypsin. Each point represents the mean ± SEM (n = 3).
Figure 3.
Discrimination of 2-fold variations in virus input.
Virus replication was assessed in the presence of TPCK-trypsin (1 µg/mL). A 2-fold dilution series of the virus was prepared (32,000 to 250 TCID50 per 100 µL per well). After incubation for 1 h at 37°C, a suspension of MDCK-London cells (30,000 in 100 µL) was added. At 6 h post-infection, experimental samples were prepared using SPR and subjected to qRT-PCR. For each virus strain, the RNA copy numbers were normalized to the mean value observed from cells infected with 250 TCID50. Each point represents the mean ± SEM (n = 3).
Figure 4.
Influenza virus microneutralization assessed by qRT-PCR (qPCR-MN).
(A) An inoculum containing 1000 TCID50 of virus (Bris/07) was mixed with a dilution from a 2-fold dilution series of ferret antiserum in a well of a 96-well plate. After allowing the neutralization reaction to proceed for 1 hour at 37°C, trypsinized MDCK-London cells (30,000 per well) were added. TPCK-trypsin was present at 1 µg/mL. After 6 hours, experimental samples were prepared using SPR and subjected to qRT-PCR. The RNA copy numbers were normalized to the mean value obtained from infected wells in the absence of neutralizing serum (virus control wells). Each point represents the mean ± SEM (n = 3). The neutralization titer was defined as the reciprocal of the highest dilution factor of serum necessary to inhibit the PCR signal by 90%. (B) Same data as in (A); however, each experimental replicate was assessed independently. The mean of these curves would result in the curve depicted in (A).
Table 1.
qPCR-MN titers determined for each experimental replicate.
Table 2.
Infectivity titers for virus stocks determined by ELISA (at 22 hours, ±TPCK-trypsin) or microscopic observation of CPE (at 72 hours, +TPCK-trypsin).
Table 3.
ELISA-MN titers determined in the absence or presence of TPCK-trypsin (n = 3 for each experiment).
Table 4.
qPCR-MN robustness assessment with respect to assay duration (±TPCK-trypsin): Bris/07.
Table 5.
qPCR-MN robustness assessment with respect to assay duration (±TPCK-trypsin): SI/06.
Table 6.
qPCR-MN robustness assessment with respect to assay duration (±TPCK-trypsin): Uru/07.
Table 7.
qPCR-MN robustness assessment with respect to input virus dose.
Figure 5.
Correlation between neutralization titers measured by qPCR-MN and ELISA-MN.
Adult human sera (n = 20) were assessed by qPCR-MN (input virus of 1000 TCID50 scored by CPE; endpoint assessment at 6 hours post-infection; +TPCK-trypsin) and ELISA-MN (input virus of 100 TCID50 scored by ELISA; endpoint assessment at 22 hours post-infection; -TPCK-trypsin). Neutralization activity against SI/06 was measured. Each point represents the log2 of the geometric mean titer derived from two experimental replicates. Certain points were nudged along the y-axis direction in order to reveal overlaps.