The amount of CVA10 particles remaining on the surface of RD cells was estimated by a one-step, quantitative RT-PCR as previously described (17). Some steps of the experimental procedure are similar to the pre- and post-attachment neutralization assay described in the above section. Briefly, CVA10 was mixed with different concentrations of mAb 2G8 either before or after the viruses were coated on preseeded cells. The mixture was then incubated at 4°C for 1 hour. After three washes with cold PBS buffer, total RNA was isolated using the QIAamp Mini Viral RNA Extraction Kit (Qiagen). RT-PCR reaction was performed with the CFX96 Real-Time PCR Detection System (Bio-Rad). Gene-specific primers (forward, 5′-TACTTTGGGTGTCCGTGTTT-3′; reverse, 5′-TGGCCAATCCAATAGCTATATG-3′; probe, 5′-FAM- AYTGGCTGCTTATGGTGACRAT-BHQ1-3′) were used for the RT-PCR experiment. The relative levels of CVA10 RNA in different samples were estimated by the comparative 2−ΔΔCt method (43).

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