The AmpC β-lactamases genes (blaCITM and blaDHAM) were amplified by PCR using plasmid DNA preparation as template. The primers used for blaCITM gene was CLR5-F (5ʹ TGG CCA GAA CTG ACA GGC AAA −3ʹ) and CLR5-R (5ʹ- TTT CTC CTG AAC GTG GCT GGC −3ʹ) as forward and reverse primer respectively while the primers for blaDHAM gene was forward sequence DHAM for (5ʹ AAC TTT CAC AGG TGT GCT GGG −3ʹ) and reverse sequence DHAM_rev (3ʹ CCG TAC GCA TAC TGG CTT TGC −5ʹ).11 (link) Reaction volume was set as 25µL by adding 12.5µL of 1X master mix (5× HOT FIREPol Blend Master Mix Ready to Load, Solis BioDyne, Estonia), 0.5µL each of the forward and reverse primers and 4µL of DNA template and ddH2O 7.5µL. The optimized PCR amplification of both the genes was 94ºC for 3 mins for initial denaturation; 35 cycles of denaturation at 94ºC for 30 seconds; 25 cycles of annealing at 62ºC for 30 seconds; 35 cycles of extension at 72º for 1 min; and final extension at 72ºC for 7 mins.11 (link),27 (link)
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Detection and Amplification of AmpC β-Lactamases
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Corresponding Organization : Tribhuvan University
Other organizations : Helen Keller International Nepal, Annapurna Neurological Institute and Allied Sciences
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Variable analysis
independent variables
- Primers used for amplification of bla_CITM gene (CLR5-F and CLR5-R)
- Primers used for amplification of bla_DHAM gene (DHAM_for and DHAM_rev)
- Amplification of bla_CITM gene
- Amplification of bla_DHAM gene
- Reaction volume (25 µL)
- Master mix (12.5 µL of 1X master mix)
- Primer concentration (0.5 µL each of forward and reverse primers)
- DNA template amount (4 µL)
- DdH2O volume (7.5 µL)
- PCR conditions (94°C for 3 mins initial denaturation, 35 cycles of 94°C for 30 s denaturation, 25 cycles of 62°C for 30 s annealing, 35 cycles of 72°C for 1 min extension, and 72°C for 7 mins final extension)
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