Gotaq green master mix protocol

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).

To ensure that patients were infected with only P. falciparum, all 263 sera were subjected to Plasmodium species characterization following genomic DNA (gDNA) isolation using QIamp DNA Mini Kit (Qiagen, Hilden, Germany) as reported by others [15 (link), 16 (link)]. DNA extracted from blood samples of known microscopically confirmed P. falciparum, P. malariae, P. vivax and P. ovale-infected patients were used as positive controls [3 (link)] to discriminate P. falciparum-positive samples from other Plasmodium-infected samples.
Qualitative detection of Plasmodium parasite DNA was based on a nested PCR approach with primers targeting the Plasmodium spp. 18S small subunit ribosomal RNA gene (18S ssrDNA) as described previously [17 (link)]. The primary and nested PCR reactions were performed using the GoTaq Green Master Mix protocol (Promega, Madison, USA), according to the manufacturer’s recommendations, and amplification conditions were as described previously [3 (link)]. Nested PCR results were scored as a categorical variable (presence versus absence of amplification).