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: Compared DNA extraction methods. The DNA extraction methods whose characteristics are compared in this study are divided into column-based, plate-based, and lysis-only methods. The hands-on time was estimated for 3 samples, and the list prices per reaction were calculated using online prices (from August 2023, Germany).

Article Snippet: column-based , NucleoSpin DNA Stool kit, Macherey-Nagel , (NS) , chemical lysis, heating, and mechanical lysis (bead beating); clean-up column; silica spin column (for binding of DNA and subsequent elution) , Isolation of human and bacterial DNA from stool samples , 50 , ≈1 h , 5.52.

Techniques: DNA Extraction, Lysis, Extraction, Binding Assay, Isolation

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 OXA-48 and bla NDM-1 DNA. The carbapenemase resistance genes bla OXA-48 and bla NDM-1 , present in K. pneumoniae strain NRZ-52799, 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 templates. 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-52799. A sample of 1 µL of eluted DNA was used as a template in qPCR assays for the detection and quantification of bla OXA-48 and bla NDM-1 . Results are shown for three independent experiments. nd: not detectable, #: inconsistent detection (only 4 of 6 PCR reactions were positive), ##: extracted DNA could not be used as a template in qPCR due to the viscosity of the samples after heat treatment. Statistical analysis was performed using Welch’s t -test. ns: not significant, *: p < 0.05; ***: p < 0.005.

Article Snippet: column-based , NucleoSpin DNA Stool kit, Macherey-Nagel , (NS) , chemical lysis, heating, and mechanical lysis (bead beating); clean-up column; silica spin column (for binding of DNA and subsequent elution) , Isolation of human and bacterial DNA from stool samples , 50 , ≈1 h , 5.52.

Techniques: DNA Extraction, Isolation, Lysis, Viscosity

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: column-based , NucleoSpin DNA Stool kit, Macherey-Nagel , (NS) , chemical lysis, heating, and mechanical lysis (bead beating); clean-up column; silica spin column (for binding of DNA and subsequent elution) , Isolation of human and bacterial DNA from stool samples , 50 , ≈1 h , 5.52.

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

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: Minimal CFU input in DNA extractions, sufficient for consistent resistance gene detection. K. pneumoniae NRZ-52799 was diluted in the range of 10 5 to 10 1 CFU/200 µL of bacterial cultures ( left ), and bacterial cultures were spiked into stool matrix ( right ) and employed for the isolation of DNA with the 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). Samples of 1 µL of isolated DNA were used as templates in the qPCR assays for the detection of bla OXA-48 , bla NDM-1 , and khe . Cultivation of K. pneumoniae NRZ-52799 and subsequent DNA extractions were performed three times in independent experiments. The isolated DNA was analyzed in duplicate in the qPCR resistance gene assays for bla OXA-48 and bla NDM-1 , and in the khe qPCR for K. pneumoniae specificity control. Black bars indicate the lowest CFU input in DNA that was sufficient for consistent qPCR results (i.e., all 6 qPCR reactions were positive). nd: not detectable, *: extracted DNA could not be used as template in qPCR due to the viscosity of the samples after heat treatment.

Article Snippet: column-based , NucleoSpin DNA Stool kit, Macherey-Nagel , (NS) , chemical lysis, heating, and mechanical lysis (bead beating); clean-up column; silica spin column (for binding of DNA and subsequent elution) , Isolation of human and bacterial DNA from stool samples , 50 , ≈1 h , 5.52.

Techniques: Isolation, Lysis, Control, Viscosity