The qPCR included primers and probe for an attenuated amplification of the RNase P gene RPPH1.
Regression curves for each of the TREC and RNase P calibrators (Fig.
2) demonstrated separation between each of the TREC calibrators when viewed alone and appropriate attenuation of the RNase P when viewed together.
Tests of DBS calibrators made from dilutions of known leukocyte concentrations into leukocyte-depleted blood (14) showed RNase P copy numbers consistent with expected concentrations of cells per milliliter (e.g., 2.6 X [10.sup.5] [+ or -] 1.9 X [10.sup.4] copies of RNase P for samples known to have 1.25 X [10.sup.5] cells/mL or 2.5 X [10.sup.5] copies RNase P; 6.3 X [10.sup.5] [+ or -] 7.1 X [10.sup.4] for samples known to have 2.5 X [10.sup.5] cells/mL; and 1.7 X [10.sup.6] [+ or -] 9.7 X [10.sup.4] for samples known to have 5.0 X [10.sup.5] cells/mL).
The TREC and RNase P values were similar to those observed with the routine DNA preparation.
The results of this investigation demonstrate the use of a multiplex qPCR assay, the Multiplex TREC qPCR, with an internal QC, the RNase P gene RPPH1, for use in population-based NBS for detection of SCID.
Although the most obvious value to the internal control is real-time validation of the quality of the sample (i.e., the DNA is amplifiable), we are investigating the analytic feasibility of using each individual sample's TREC and RNase P ratio to control for sampling error within the filter-paper matrix.