Eva green 1x

PCR amplification of the tcdA, tcdB, and cdtB genes was performed in 25 μl reaction with GoTaq Green Master Mix (Promega, United States), using the primers described in Table 1. Thermocycler conditions were 15 min at 95°C, followed by 30 cycles of 30 s at 94°C, 90 s at 57°C and 60 s at 72°C, with a final extension of 7 min at 72°C. We also designed primers to amplify the complete tcdC gene (Table 1) using the following conditions, 5 min at 94°C, followed by 30 cycles of 1 min at 94°C, 1 min at 42.5°C and 1 min at 72°C, with a final extension of 4 min at 72°C. Products were analyzed by 2.0% agarose electrophoresis, stained with Eva Green 1X (Jena Bioscience, Germany). Additional genotyping of the strains was done by ribotyping as described previously (Bidet et al., 1999 (link)). DNA from a clinical C. difficile strain ribotype 027 (Kindly donated by Dra. Elvira Garza, Monterrey, Mexico) (Camacho-Ortiz et al., 2015 (link)) was included in each run as a reference. Amplification products were subjected to electrophoresis in a 2.5% agarose gel, ethidium bromide stained and analyzed under ultraviolet light using the LabWorks Image Acquisition and Analysis Software (Version 4.5.00.0 for Windows, UVP).

All samples were obtained from patients suffering from hospital-acquired diarrhea, attended at the National Medical Center “XXI Century” of the Mexican Institute of Social Security, Mexico City. Stools were cultured on CCFA agar after an ethanol shock and incubated under anaerobic conditions (N₂, 5% CO₂, and 5% H₂) for 48–72 h at 37°C. Isolates were purified by passages of single colonies on Casman Blood Agar (Difco; Becton, Dickinson and Company, United States) and CCFA agar at least three times. C. difficile strains were identified by Gram staining, morphological growth in blood agar, UV-fluorescence and PCR amplification of 16S rRNA and tpi genes. Isolates were also subcultured in meat broth (Difco; Becton, Dickinson and Company, United States) for storage.
The amplification of 16S rRNA gene was performed using primers PS13 and PS14 (Table 1), while tpi gene amplification was performed with tpi-Fw and tpi-Rv primers in a 25 μl reaction according to the GoTaq Green Master Mix Protocol (Promega, United States). Thermocycler conditions were 7 min at 95°C, followed by 30 cycles of 30 s at 94°C, 90 s at 57°C and 1 min at 72°C; then a final extension of 7 min at 72°C. Products were analyzed by 2.0% agarose electrophoresis stained with Eva Green 1X (Jena Bioscience, Germany).