Table 1.
Primers and probes used in this study.
Fig 1.
Internal reference control (IRC) duplex RT-PCR assay format.
The target and IRC templates share the same primer set, the specific probes for the target and IRC are labeled with different fluorescent dyes, and the assay is performed in one reaction.
Fig 2.
Multiple sequence alignment of the universal 16S primers and probes with the 16S rDNA sequences.
The 16S rDNA sequences were obtained from 28 bacterial species (their names and NCBI Gene IDs are shown on the left of the alignment).
Fig 3.
Multiple sequence alignment of the universal 23S primers and probes with the 23S rDNA sequences.
The 23S rDNA sequences were obtained from 28 bacterial species (their names and NCBI Gene IDs are shown on the left of the alignment).
Fig 4.
Specificity testing of the 16S/23S primers.
PCR and melt curve analyses were used to detect nine bacterial strains and evaluate the specificity of the 16S/23S primers.
Fig 5.
Internal reference control (IRC) duplex RT-PCR: testing for reliability, competition, and inhibition.
The copy number of the IRC template was held at five copies, whereas the E. coli gDNA was titrated from two copies to 50 copies. A: IRC-16S duplex RT-PCR assay. B: IRC-23S duplex RT-PCR assay.
Table 2.
Sensitivity, linearity, and efficiency of internal reference control (IRC) duplex RT-PCR.
Table 3.
Internal reference control (IRC) duplex RT-PCR testing of human platelet-rich plasma spiked with E. coli.
Table 4.
Results of the spiking study in which the detectability of 16S/23S was tested using internal reference control (IRC) duplex RT-PCR.