A RevertAid TM H Minus Reverse Transcriptase kit (Fermentas) was used to synthesize cDNA according to the manufacturer's instruction. Random hexamers were used to convert 750 ng RNA into cDNA. The synthesized cDNA was treated with RNase (Qiagen) for 1 hour at 37uC and stored at 220uC. The software program Molecular Beacon (Premier Biosoft) was used to design primer pairs and probes (Table 2) for the 2 multiplex quantitative PCRs (qPCR), based on the available genome of C. difficile strain 630 [35] . All primer pairs were first tested by conventional PCR and multiplex PCR to confirm specificity and amplicon sizes. The primer pair and the probe for the amplification of the tcdC gene are in front of the insertion site in the tcdC gene ( Figure 1A ), allowing detection of tcdC transcription levels in wild type and CT::tcdC strains. The real-time multiplex qPCR amplification of the PaLoc genes and the reference gene encoding for a ribosomal protein (rpsJ) was performed on a CFX96 real-time PCR detection system (Biorad) [31] . The amplification efficiencies of the PaLoc and reference genes were determined using serially diluted genomic DNA (standard curve). The manually calculated efficiencies and the reference gene rpsJ were used to normalize the expression levels of the PaLoc genes. The amplification was performed in a 25 ml final volume. The first real-time multiplex qPCR (target genes: tcdA, tcdA and tcdC) contained 25 ml Hotstar mastermix (Qiagen), forward and reverse primers (80 nm each primer), 2.5 mM MgCl 2 , 100 nM of each probe and 2 ml synthesized cDNA. The second multiplex real-time multiplex Q-PCR (target genes: tcdR, tcdE) contained 25 ml Hotstar mastermix (Qiagen), forward and reverse primers (80 nm each primer), 3.5 mM MgCl 2 , 100 nM of each probe and 2 ml synthesized cDNA. The real-time qPCR to quantify the reference gene rpsJ contained 25 ml Hotstar mastermix (Qiagen), forward and reverse primers (80 nm each primer), 2.5 mM MgCl 2 , 0.06% SYBRgreen (Sigma) and 2 ml synthesized cDNA. The real-time qPCR protocol included an enzyme activation step for 15 min at 95uC, followed by 50 cycles of amplification; 95uC for 30 sec, 52uC for 30 sec and 72uC for 30 sec.