Evaluation of consistency in quantification of gene copy number by real-time reverse transcription quantitative polymerase chain reaction and virus titer by plaque-forming assay for human respiratory syncytial virus.

Abstract

The plaque-forming assay is the standard technique for determining viral titer, and a critical measurement for investigating viral replication. However, this assay is highly dependent on experimental technique and conditions. In the case of human respiratory syncytial virus (RSV) in particular, it can be difficult to objectively confirm the accuracy of plaque-forming assay because the plaques made by RSV are often small and unclear. In recent studies, RT-qPCR methods have emerged as a supportive procedure for assessment of viral titer, yielding highly sensitive and reproducible results. In this report, we compare the viral replication, as determined by plaque-forming assay, and the copy numbers of RSV genes NS1, NS2, N, and F, as determined by RT-qPCR. Two real-time PCR systems, SYBR Green and TaqMan probe, gave highly similar results for measurement of copy numbers of RSV N genes of virus subgroups A. We determined the RSV gene copy numbers in the culture cell supernatant and cell lysate measured at various multiplicities of infection. We found that copy number of the RSV N gene in the culture supernatant and cell lysate was highly correlated with plaque-forming units. In conclusion, RT-qPCR measurement of RSV gene copy number was highly dependent on viral titer, and the detailed comparison between each gene copy number and virus titer should be useful and supportive in confirming RSV plaque-forming assay and virus dynamics. The technique may also be used to estimate the amount of RSV present in clinical specimens.