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[…] AM, 6-carboxylfluorescein) and the unlabeled reverse primer 5′-GTGCCAGCAAGATCCAATCTAGA-3′, was added. PCR amplification was performed using 35 cycles of 95°C for 30 s, 60°C for 30 s, and 72°C for 45 s, followed by 72°C for 20 min. The resulting MLPA amplicons were examined by agarose gel (3%) electrophoresis and by fragment analysis using an ABI 3130xl capillary electrophoresis system (Applied Biosystems). For fragment analysis, 0.5 μl of PCR amplicon was mixed with 9.25 μl of Hi-Di formamide and 0.25 μl 500 LIZ size standard (GeneScan; Applied Biosystems). The mixture was incubated for 3 min at 95°C, chilled for 10 min, and analyzed. The resulting fragment analysis data were analyzed using GeneMapper v4.0 (Applied Biosystems). Data were analyzed using BioNumerics (Applied Maths, Belgium), and phylogenetic trees were drawn using Dendroscope v2.3., Probes comprise left and right hybridization sequences., Probe contains a nucleotide to detect a specific SNP in the QRDR region (boldface and underlined)., gyrA probe requires a spanning oligonucleotide to detect mutations at codons 83 and 87 (TGGTGTCATACACTGCGA)., A cross-sectional subset of 73 S. Typhi isolates was arbitrarily selected to encompass a range of locations, antimicrobial resistance patterns, studies in Vietnam, and dates of isolation for gold-standard SNP genotyping using the Illumina GoldenGate platform (see Table S1 in the supplemental material). DNA was extracted from all S. Typhi isolates as described above, and concentrations were assessed using the Qu […]

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