Figure 1
Schematic representation of the single-tube nested real-time PCR amplification and sequences of primers/probes. (a) Schematic showing how the single-tube nested qPCR works. At the first round, the PCR was performed to amplify a 428-bp fragment with an external primer set (NF6110 and NR6110). The larger amplicon produced by the first round of PCR was used as a template for the second round of PCR. The second pair of primers and probe (IS4F, IS4R and IS4P) bind within the first round of amplicon to produce a fragment shorter in length (141-bp). Second round amplicons were detection with a FAM-labeled probe (495–520 nm). The internal control (Lambda phage DNA) is only co-amplified with target DNA (IS6110-containing vector) with Ld2F, Ld2R and RPLd at the second round of PCR and the products were detection with a Cy5-labeled probe (646-662 nm). The PCR program of the assay was shown in the right-hand side. (b) The nested-qPCR amplicon of IS6110 from MTBC and Lambda nucleotide sequences are shown. The sense primers were shown as black letters on a purple background and the anti-sense primers were indicated as black letters on an orange background.
