Intra- and inter-assay variability of the 16S rRNA RT/IS2404 qPCR assay.

Table 5 shows intra- and inter-assay variability of the 16S rRNA RT/IS2404 qPCR assay.16S rRNA RT-qPCR: 16S rRNA gene standards (935 bp) were generated by conventional PCR according to Talaat et al. [12]. Quantification of PCR products was conducted by Picogreen fluorometry (Invitrogen) and copy numbers were calculated based on the known mass of one amplicon. Serial standards were prepared from PCR products in 5 Log dilutions ranging from 3E+6 (standard no. 1) to 3E+2 copies (standard no. 5) of the 16S rRNA amplicon (PCR template: 2 µl) and were subjected to the 16S rRNA RT-qPCR in quadruplicate on one 96-well plate to assess intra-assay variability. The runs were repeated on three days to determine the inter-assay variability between runs 1 through 3. The intra- and inter-assay variability of the 16S rRNA RT-qPCR was low with maximum coefficients of variation (CV) of 0.49 (intra-assay) and 1.33 (inter-assay).IS2404 qPCR: Cloned IS2404 replicates (1,047 bp, complete sequence; M. ulcerans Agy99) were used as standards. Quantification of IS2404 templates was conducted by Picogreen fluorometry (Invitrogen) and copy numbers were calculated based on the known mass of one template. Serial standards were prepared in 7 Log dilutions ranging from 2E+8 to 2E+2 copies of the IS2404 (PCR template: 2 µl) and were subjected to the IS2404 qPCR in quadruplicate on one 96-well plate to assess intra-assay variability. The runs were repeated on three days to determine the inter-assay variability between runs 1 through 3. The intra- and inter-assay variability of the IS2404 qPCR was low with maximum CV of 0.65 (intra-assay) and 2.66 (inter-assay).a16S rRNA, target of the 16S rRNA RT-qPCR; IS2404, target of the IS2404 qPCR.bCt-range, range of Ct-values of samples tested in the same dilution.cCV, coefficient of variation of copy numbers from samples tested in quadruplicate of the same dilution.dΔCt max., maximum Ct-variation of all samples tested within one run.eCV max., maximum CV of all samples tested within one run.fCt-range, range of Ct-values of samples tested in the same dilution within three runs.gCV of samples in the same dilution tested within three runs.hΔCt max. of all samples tested within three runs.iCV max. of all samples tested within three runs.