Dynamic range and limit of detection of the DENV RT-PCR assay.

<p>(A) The linear dynamic range of the DENV RT-PCR assay was determined by testing triplicates of 10-fold serially diluted in vitro transcribed RNA. The sequences of the transcript RNA (RNA[DENV_R1–3] and RNA[DENV_R4]) were matched with the two reverse primers DENV_R1–3 and DENV_R4, respectively. Each dot represents the mean Cq-value from three replicates, the error bars indicate the 95% confidence interval, and the lines illustrate the result of the lin-log regression analysis. (B and C) Limit of detection was determined by assaying eight replicates of twofold serially diluted RNA transcripts in three separate experiments, and the results of testing are shown for RNA[DENV_R1–3] (B) and RNA[DENV_R4] (C). Horizontal lines indicate mean values, boxes denote the 25th to 75th percentiles and whiskers the 5–95% percentiles, and dots represent outliers. The number of positives per total number of replicates tested is given above each box. Limit of detection was defined as the last dilution in which transcript RNA was detected in all 24 replicates. GCE = genome copy equivalents.