The MST was created based on the categorical coefficient and a priority rule consisting of the highest number

of single-locus variants. Cilengitide The polymorphism index of individual or combined VNTRs was calculated using the Hunter-Gaston discriminatory index (HGDI) [23]. Results Identification of VNTRs for MLVA typing Among the 130 TRs tested, only five were polymorphic with different allele sizes, making them useful for discriminating among types. The five VNTRs selected are distributed around the genome from nucleotide positions 181200 to 298794 in the M. hominis PG21 reference strain (Table 1). The PCR products ranged in size from 153 to 290 bp in the M. hominis KPT-8602 order PG21 reference strain. All of the VNTRs were located in open reading frames (ORFs). Markers Mho-52, Mho-53 and Mho-116 were located in the rpoD gene encoding the RNA polymerase sigma factor RpoD, the pgsA gene encoding the CDP-diacylglycerol-glycerol-3-phosphate-3-phosphatidyl transferase and the oppA gene encoding the oligopeptide ABC transporter substrate-binding protein, respectively. The two other markers were located in ORFs encoding hypothetical proteins. The sizes of the unit repeats ranged from 3 bp to 42 bp.

Sequencing the PCR products of different sizes at each of the five loci from each of the 12 screening isolates confirmed the sizes and sequences of the individual VNTR loci. Table 1 Characteristics of the five VNTR markers Name Nucleotide positiona(bp) Locus (protein no. in the genome sequence) Repeat size (bp) Consensus sequence % identity between VNTRs HGDIb Mho-50 298627-298794 Hypothetical protein, predicted lipoprotein (MHO_2440) 42 TCAAGATTCTACAACCACAGGTGAAGATTCGACTGGACAATC 98 0.313 Mho-52 259317-259340 rpoD gene (MHO_2150) 3 GAT 82 0.203 Mho-53 246308-246325 pgsA gene (MHO_2070) 3 ATT 100 0.784 Mho-114 190335-190346 Hypothetical protein, predicted lipoprotein (MHO_1590) 6 TTGGCT Acetophenone 100 0.336 Mho-116 181200-181202 oppA gene (MHO_1510) 3 GAA 100

0.020 aPosition (5’ end) on the M. hominis PG21 genome sequence. bHGDI, Hunter Gaston Diversity Index. The stability of the five polymorphic markers in five strains was examined after 10 serial passages in Hayflick modified broth medium supplemented with arginine. The analysis of the five strains resulted in identical MLVA profiles for all markers. The use of fluorescently labelled primers in two multiplex PCRs (Mho-50, Mho-52 and Mho-53 for PCR T1 and Mho-114 and Mho-116 for the PCR T2), and capillary electrophoresis facilitated the interpretation of the results, an improvement over the standard agarose gel electrophoresis. Using GeneMapper Software, all loci were clearly identified on electropherograms according to their size ranges and colours, and the amplicon sizes allowed the determination of repeat number.