Detecting Beta-Lactamase Genes by PCR

DNA extraction was performed using a QIAamp® DNA Mini kit (QIAGEN, Hilden, Germany) following the manufacturer’s instructions. The molecular detection of beta-lactamases (bla_CTX-M-1, bla_CTX-M-2, bla_CTX-M-9, bla_CTX-M8/25, bla_TEM and bla_SHV) was performed by end-point PCR using previously reported primers [31 (link),32 (link)]. The reaction was performed in a GenAmp ® PCR System 9700 (Applied Biosystems). The final volume of the reaction mixture was 25 μL and contained 12.5 μL of AmpliTaq Gold® 360 MasterMix (Applied Biosystems, Foster City, CA, USA) and 0.4 pmol/μL of the primers. The obtained fragments were purified and sequenced in an ABI Prism 310 analyzer. The sequences were analyzed with nBLAST program [33 (link),34 (link)], and analyzed using multiple alignments with the BioEdit program v7.2.5.0 (Ibis Biosciences, Carlsbad, CA, USA) to determine the beta-lactamase subtype.