The linear dynamic range of the rtLC DBS assay was initially assessed using a 5 log 10 dilution series of the customized standard DBS. The assay was shown to be linear over the entire range of 586,000 to 37 copies. A linear regression of the rtLC DBS customized standards copies on true concentrations yielded a correlation coefficient of 0.984 (P,0.001) and the fitted model is shown in Figure 1 . The slope of 1.037 closely approximates the theoretical maximum amplification efficiency of 100%; the fitted slope is slightly greater than 1 due to the several undetected cases in low range. To estimate the detection limit of the assay, we performed 22 extra runs at low concentrations (7, 17, 37, 83, 187 copies), which spread evenly in log scale and contained two concentration 37 and 187 that were already in design. We used all available data at concentrations from 7 to 937 to fit the probit model to get estimations for 95% detection limit as 135, with 95% confidence interval [82, 223]; 99% detection limit was estimated as 292, with 95% confidence interval [147, 583] ( Table 2) .