bla kpc 2 nucleotides 3326 4209 from genbank reference sequence nz cp023480 1  (Thermo Fisher)

Journal: Microorganisms

Article Title: Detection of Klebsiella pneumoniae Carbapenem Resistance Genes by qPCR: Choosing the Right Method for Total DNA Extraction

doi: 10.3390/microorganisms12071285

Figure Lengend Snippet: Analytical sensitivity of the qPCR assays. The newly established qPCRs for the detection of bla OXA-48 , bla NDM-1 , bla KPC-2 , and bla VIM-1 were validated with the help of synthetic DNA standards. Dilutions ranging from 10 0 to 10 7 DNA copies/reaction for the DNA standards of bla OXA-48 (blue), bla NDM-1 (green), bla KPC-2 (orange), and bla VIM-1 (violet) were employed in qPCR reactions. Eight PCR reactions were performed for each DNA concentration. Upper panel : Fluorescence signals of the qPCR reactions. Each set of eight curves represents one DNA concentration of the decadic dilution series ranging from 10 7 ( left ) to 10 0 ( right ). Middle panel : Standard curves calculated by linear regression indicating constant performance of the qPCR assays in the tested concentration range. Lower panel : The limits of detection (LOD, with 95% probability), calculated by probit analysis , for each resistance gene assay. bla: beta-lactamase, NDM: New Delhi metallo-β-lactamase, OXA: oxacillinase β-lactamase, KPC: K. pneumoniae carbapenemase, VIM: verona integron-encoded metallo-β-lactamase, C t : cycle threshold, RFU: relative fluorescence unit(s).

Article Snippet: Sequences of bla KPC-2 (nucleotides 3326–4209 from GenBank reference sequence NZ_CP023480.1) and bla VIM-1 (nucleotides 101–901 from GenBank reference sequence NG_050336.1) were synthesized and inserted into the vectors pMA-RQ and pMA-T, respectively (Thermo Fisher Geneart, Regensburg, Germany).

Techniques: Bla VIM Assay, Concentration Assay, Fluorescence, Gene Assay

Journal: Microorganisms

Article Title: Detection of Klebsiella pneumoniae Carbapenem Resistance Genes by qPCR: Choosing the Right Method for Total DNA Extraction

doi: 10.3390/microorganisms12071285

Figure Lengend Snippet: Effect of DNA extraction methods on quantitative detection of bla KPC-2 and bla VIM-1 DNA. The carbapenemase resistance genes bla KPC-2 and bla VIM-1 , present in K. pneumoniae strain NRZ-43730, were detected by qPCR using DNA isolated with NucleoSpin DNA Stool kit (NS, red), QIAamp Fast DNA Stool Mini kit (QS, orange), EchoLUTION Buccal Swab DNA Kit (BE, yellow), EchoLUTION Viral RNA/DNA Swab Kit Plus (BV, green), Quick Extract Solution (QE, blue), Thermal lysis P (TL-P, violet), and Thermal lysis T (TL-T, pink) as template. DNA was isolated either from bacterial culture samples ( upper panel ) or from stool matrix spiked with bacterial culture samples ( lower panel ) each containing 10 5 CFU K. pneumoniae strain NRZ-43730. Samples of 1 µL of eluted DNA were used as templates in qPCR assays for the detection and quantification of bla KPC-2 and bla VIM-1 . Results are shown for three independent experiments. nd: not detectable, ##: extracted DNA could not be used as template in qPCR due to the viscosity of the samples after heat treatment. Statistical analysis was performed by Welch’s t -test. ns: not significant, *: p < 0.05; **: p < 0.01; ***: p < 0.005.

Article Snippet: Sequences of bla KPC-2 (nucleotides 3326–4209 from GenBank reference sequence NZ_CP023480.1) and bla VIM-1 (nucleotides 101–901 from GenBank reference sequence NG_050336.1) were synthesized and inserted into the vectors pMA-RQ and pMA-T, respectively (Thermo Fisher Geneart, Regensburg, Germany).

Techniques: DNA Extraction, Bla VIM Assay, Isolation, Lysis, Viscosity