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Rapid detection of the New Delhi metallo-b-lactamase 1 (NDM-1) gene by loop-mediated isothermal amplification (LAMP).

ABSTRACT: BACKGROUND:New Delhi Metallo-b-lactamase (NDM-1) is an enzyme emerging around the world conferring resistance to a wide range of ?-lactams agents and whose early detection is extremely important. We proposed to standardize the detection of the blaNDM-1 gene using the LOOP-mediated isothermal amplification technique (LAMP). METHODS:In all, 14 Gram-negative bacterial strains isolated from patients presenting pneumonia associated with mechanical ventilation were used for the blaNDM-1 standardization by LAMP. Klebsiella pneumoniae ATCC BAA-2473 and two clinical strains were used as a positive control. All results were compared to the reaction in polymerase chain reaction (PCR), considered gold standard for this detection. RESULTS:There was an excellent correlation between the two techniques employed, since all measured clinical strains were negative in both employed tests and two clinical, and a reference strains were positive. CONCLUSIONS:The lamp technique seems to be an excellent option for the rapid detection of blaNDM-1. The amplification time is much shorter than other molecular techniques, the PCR machine is not necessary, it is easy of implementation and costs is low.

SUBMITTER: Moreira MG

PROVIDER: S-EPMC6816916 | biostudies-literature | 2018 May

REPOSITORIES: biostudies-literature

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Rapid detection of the New Delhi metallo-b-lactamase 1 (NDM-1) gene by loop-mediated isothermal amplification (LAMP).

Moreira Mirna Giselle MG Barreto Lidia Miranda LM Dos Santos Vera Lúcia VL Monteiro Andrea Souza AS Nobre Vandack V Dos Santos Simone Gonçalves SG

Journal of clinical laboratory analysis 20170928 4

<h4>Background</h4>New Delhi Metallo-b-lactamase (NDM-1) is an enzyme emerging around the world conferring resistance to a wide range of β-lactams agents and whose early detection is extremely important. We proposed to standardize the detection of the bla<sub>NDM</sub><sub>-1</sub> gene using the LOOP-mediated isothermal amplification technique (LAMP).<h4>Methods</h4>In all, 14 Gram-negative bacterial strains isolated from patients presenting pneumonia associated with mechanical ventilation were ...[more]

PMID: 28960568

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Project description:BackgroundBlaAFM-1 (GenBank Accession No. 143105.1) is a new B1 subclass metallo-β-lactamase gene discovered by our group, and isolated from an Alcaligenes faecalis plasmid that renders carbapenem antibiotics ineffective. In this study, we generated a fast and reliable assay for blaAFM-1 detection.MethodsWe designed optimum loop-mediated isothermal amplification (LAMP) primers and constructed a recombinant plasmid AFM-1 to specifically detect blaAFM-1. Optimal LAMP primers were used to assess sensitivity of the recombinant plasmid AFM-1 and blaAFM-1-supplemented samples (simulated sputum and simulated feces). Fifty two samples, without blaAFM-1, were used to assess LAMP real-time assay specificity; these samples were verified by conventional PCR and sequencing for the absence of blaAFM-1. Three hundred clinical Gram-negative carbapenem-resistant strains were tested by LAMP assay for strains carrying blaAFM-1, which were confirmed by conventional PCR and Sanger sequencing. We calculated the sensitivity and its 95% confidence interval (95% CI), specificity and its 95% CI, and predictive values of the LAMP assay and conventional PCR/sequencing by investigating positive and negative clinical strains.ResultsThe lowest limit of detection for the recombinant plasmid AFM-1 and blaAFM-1-supplemented samples (in both simulated sputum and simulated feces) was 101 copies/reaction. All amplification curves of the 52 blaAFM-1-free bacteria strains were negative, suggesting the LAMP assay had excellent specificity for detecting blaAFM-1. Among the 300 clinical strains, eight were positive for blaAFM-1 using LAMP. These LAMP results were consistent with conventional PCR and Sanger sequencing data. As with conventional PCR/sequencing, the LAMP method exhibits 100% sensitivity (95% CI 59.8-100%) and 100% specificity (95% CI 98.4-100%) for blaAFM-1 detection. The LAMP assay is also time-efficient (1 h) for blaAFM-1 detection.ConclusionsWe established a new LAMP assay with high sensitivity and specificity to detect the novel B1-β-lactamase gene, blaAFM-1.

Dataset Information

Rapid detection of the New Delhi metallo-b-lactamase 1 (NDM-1) gene by loop-mediated isothermal amplification (LAMP).

Publications

Rapid detection of the New Delhi metallo-b-lactamase 1 (NDM-1) gene by loop-mediated isothermal amplification (LAMP).

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