PCR amplification of toxin genes was performed to detect the toxin genes tcdA, tcdB, cdtA and cdtB (Table 6 ), using the following primer pairs. The primers NK 2 (5′-CCCAATAGAAGATTCAATATTAAGCTT-3′) and NK 3 (5′-GGAAGAAAAGAACTTCTGGCTCACTCAGGT-3′) served to amplify the non-repetitive region of tcdA, NK 9 (5′-CCACCAGCTGCAGCCATA-3′) and NK 11 (5′-TGATGCTAATAATGAATCTAAAATGGTAAC-3′) the repetitive region of tcdA. For tcdB, the primer pair NK 104 (5′-GTGTAGCAATGAAAGTCCAAGTTTACGC-3′) and NK 105 (5′-CACTTAGCTCTTTGATTGCTGCACCT-3′) was used [52 (link),53 (link)]. For the binary toxin, primers cdtApos (5′-TGAACCTGGAAAAGGTGATG-3′) and cdtArev (5′-AGGATTATTTACTGGACCATTTG-3′) were used to amplify cdtA; cdtBpos (5′-CTTAATGCAAGTAAATACTGAG-3′) and cdtBrev (5′-AACGGATCTCTTGCTTCAGTC-3′) for cdtB [54 (link)].
PCR ribotyping was performed with capillary gel electrophoresis according to a modified version of the protocol of Indra et al. [55 (link)]. Primers 16S 6FAM (5′-6FAM-GTGCGGCTGGATCACCTCCT-3′) and 23S (5′-PET-CCCTGCACCCTTAATAACTTGACC-3′) were used. DNA was diluted to 1 ng/µL and 1 µL was added to 24 µL of a PCR-mix containing 0.2 µL (5 U/µL) DreamTaq DNA polymerase (Thermo Fisher Scientific, Waltham, MA USA), 1 µL of each primer (working dilution of 10 pmol/µL), 1 µL of 10 mM dNTP-Mix (Carl Roth, Karlsruhe, Germany), 1 µL MgCl2 (25 mM) (Qiagen, Hilden, Germany), 2.5 µL DreamTaq buffer (Thermo Fisher Scientific, Waltham, MA USA) and 17.3 µL water.
PCR was performed as follows: 95 °C (2 min) for initial denaturation; 30 cycles of 95 °C (30 s) for denaturation, 52 °C (30 s) for annealing, 72 °C (2 min) for elongation; 72 °C (8 min) for the final extension. PCR products were diluted 1:75 with water and ribotyping was performed by capillary electrophoresis followed by a web-based analysis. The fragment separation was performed with a SeqStudio Genetic Analyzer, SeqStudio™ Cartridge v2 (POP-1 polymer, four capillaries, 28 cm capillary length) and GeneScan™ 600 LIZ™ Size Standard v2.0. The conditions of separation were defined via FragAnalysis Run Module. The fragment length results were converted into a suitable data format and analysed with the publicly accessible WEBRIBO database to assign a ribotype [56 ].
PCR ribotyping was performed with capillary gel electrophoresis according to a modified version of the protocol of Indra et al. [55 (link)]. Primers 16S 6FAM (5′-6FAM-GTGCGGCTGGATCACCTCCT-3′) and 23S (5′-PET-CCCTGCACCCTTAATAACTTGACC-3′) were used. DNA was diluted to 1 ng/µL and 1 µL was added to 24 µL of a PCR-mix containing 0.2 µL (5 U/µL) DreamTaq DNA polymerase (Thermo Fisher Scientific, Waltham, MA USA), 1 µL of each primer (working dilution of 10 pmol/µL), 1 µL of 10 mM dNTP-Mix (Carl Roth, Karlsruhe, Germany), 1 µL MgCl2 (25 mM) (Qiagen, Hilden, Germany), 2.5 µL DreamTaq buffer (Thermo Fisher Scientific, Waltham, MA USA) and 17.3 µL water.
PCR was performed as follows: 95 °C (2 min) for initial denaturation; 30 cycles of 95 °C (30 s) for denaturation, 52 °C (30 s) for annealing, 72 °C (2 min) for elongation; 72 °C (8 min) for the final extension. PCR products were diluted 1:75 with water and ribotyping was performed by capillary electrophoresis followed by a web-based analysis. The fragment separation was performed with a SeqStudio Genetic Analyzer, SeqStudio™ Cartridge v2 (POP-1 polymer, four capillaries, 28 cm capillary length) and GeneScan™ 600 LIZ™ Size Standard v2.0. The conditions of separation were defined via FragAnalysis Run Module. The fragment length results were converted into a suitable data format and analysed with the publicly accessible WEBRIBO database to assign a ribotype [56 ].
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