1 μM primer set, and 1 U Taq DNA polymerase (BioVan, Taiwan). The PCR cycle conditions were as follows: 94°C for 5 min, followed by 30 cycles of 94°C for 40 s, annealing #Saracatinib randurls[1|1|,|CHEM1|]# temperature for 90 s, and 72°C for 50 s, and a final extension at 72°C for 3 min. Fragment analysis of the multiplex PCR products was performed as follows: 1 μL of each 20-fold-diluted PCR product,
0.1 μL GeneScan 500 LIZ size standard (Applied Biosystems, Warrington, UK) and 8.9 μL HiDi (Applied Biosystems, Foster, CA) were mixed and denatured at 95°C for 5 min. The products were then analyzed on an ABI3130 sequence detection system (Applied Biosystems). The obtained fragment sizes were exported as an Excel spreadsheet file (Microsoft, Redmond, WA). The corresponding www.selleckchem.com/products/pf299804.html copy numbers were calculated by comparison to the size of reference strains using Excel software (Microsoft). The equation used for calculation of copy number is as follows: Copy number of VNTRn = [(Fs-Fr)/repeat size of VNTRn] + copy number of reference strain, where Fs, fragment size of test strains in each VNTR loci; Fr, fragment size of reference in each VNTR loci; VNTRn, either locus
in 40 VNTR loci. Capillary gel electrophoresis-based PCR ribotyping Genomic DNA from all the C. difficile strains was amplified with the primer set designed by Bidet et al. [18], and the electrophoresis-based PCR-ribotyping was performed using a method modified from Indra et al. [19]. Briefly, the primer was labeled with carboxyfluorescein (FAM) dye to enable DNA sequence analysis. The PCR mixture included the following reagents: 25 ng genomic DNA, 1 μL buffer (10 mM Tris-HCl [pH 8.3], 50 mM
KCl, and 1.5 mM MgCl2; BioVan, Taiwan), 200 μM dNTPs, 1.5 mM MgCl2, and 1 U Taq polymerase (BioVan, Taiwan) in a 20 μL final volume. One microliter of each 20-fold-diluted PCR product, 0.8 μL Genflo625 ROX-labelled DNA Ladder (Chimerx, USA), and 8.2 μL HiDi (Applied Biosystems, Foster, CA) were mixed and denatured at 95°C for 5 min and then analyzed with a ABI3130 sequence detection system. The ribotype fragments for the full-length sequencing of strain NCTC13307 (C. difficile 630) were first predicted by the PCR-amplification function from in silico analysis using second the website (http://insilico.ehu.es), and the curve file from the ABI sequencer was confirmed by the predicted size. Ribotypes 001, 012, 017, 027, and 106 were set up by comparing the curve files with the five reference strains NCTC11204, NCTC13307, NCTC13366, NCTC 13287, and NCTC13404, respectively. All PCR-ribotypes were named with an “”R”" prefix before the serial number. Allelic diversity and typeability measurement The allelic diversity of each VNTR locus was measured by its Simpson’s index [41] and confidence interval (CI) [42]. The ability of each VNTR locus to type the 142 isolates was measured as follows: Number of isolates amplified in each VNTR locus/142.