00 [41, 42]. For 36 of these repeat regions, it was possible
to design PCR primers targeting flanking sequences, and from 28, PCR amplification products could reliably be generated from a panel of reference isolates. However, at 25 of these loci, sequence variation was insufficient to discriminate widely distributed strains, including ribotypes 027, 017, and 001 (not shown). The remaining three repeat regions could discriminate most of the ribotypes examined. The two most variable loci were designated TR6 and TR10 (Table 1). They are located at positions 0.7 Mb and 3.7 Mb of the C. difficile 630 chromosome, respectively, and exhibited Selleck FRAX597 both, sequence and length polymorphisms. Locus TR6 is composed of 21-basepair repeat AZD1480 in vivo units and resides within an open reading frame encoding a hypothetical protein (orf CD0603 in the 630 genome sequence). A homology search in public databases did not identify any significant similarities with known proteins. In Bucladesine chemical structure contrast, TR10 is located within a predicted non-coding region. It consists of 22-basepair repeats. Table 1 Characteristics of tandem repeat loci TR6 and TR10. tandem repeat locus Locationa Size (bp) Copy no. Rangeb No. of different repeatsb Repeat consensus TR6 725321 : 725600 21 7–37
80 CTTGCATACCACTAATAGTGC TR10 3753166 : 3753574 22–23 4–26 51 AAATTAATTATTATATTTCTTT a Genome location based on C. difficile 630 sequence http://www.sanger.ac.uk.
b Based on analysis of 154 isolates typed in this study. We developed a DNA based typing scheme for C. difficile based on the sequence variation of TR6 and TR10. To facilitate the application of the tandem repeat sequence typing (TRST) scheme, a duplex PCR was designed which allowed simultaneous amplification of both loci (Figure 1). Sequence data were generated from duplex PCR products using the same primers as for amplification. Nucleotide sequences from TR6 and TR10 were concatenated and unique repeat successions were assigned distinct TRST types (tagged with consecutive numbers, prefixed with “”tr”"; Figure 2, Additional files 1, 2). A detailed comparison of TRST PLEKHM2 with PCR ribotyping is described in the following. Figure 1 Results from duplex PCR amplification of loci TR6 and TR10, performed on isolates representing various ribotypes as indicated. S, 100 bp DNA ladder; N, negative control; isolates (ribotypes): VPI10463 (087); 630 (012); NCTC 13366 (027); TR13 (005); N485 (042); SMI055 (066); NCTC 11204 (001); FR535 (150); FR505 (032). Figure 2 Phylogenetic analysis (neighbor joining) based on the repeat successions in concatenated TR6 and TR10 sequences from 154 C. difficile isolates.