Development of a Novel Loop-Mediated Isothermal Amplification Method to Detect Guiana Extended-Spectrum (GES) β-Lactamase Genes in Pseudomonas aeruginosa

Abstract

Infections caused by multidrug-resistant Pseudomonas aeruginosa in hospitalized patients are often fatal, and nosocomial infections caused by Guiana extended-spectrum (GES) β-lactamase-producing strains are of growing concern. Several genotypes of the GES β-lactamase gene (blaGES) include a single missense mutation, a change from G to A at nucleotide position 493 (G493A) that changes glycine to serine; the mutant enzyme exhibits carbapenemase activity. Rapid and reliable identification of drug-resistance is important in clinical settings; however, culture methods remain the gold standard. Conventional and real-time PCR cannot identify carbapenemase-producing genotypes, and direct DNA sequencing is essential. We established a novel loop-mediated isothermal amplification (LAMP) method to detect various genotypes of blaGES and another LAMP method to discriminate carbapenemase genotypes of blaGES. We evaluated the two assays using clinical P. aeruginosa strains. Two primer sets targeting blaGES (GES-LAMP) and the point mutation (Carba-GES-LAMP) were designed and evaluated for specificity and sensitivity. The detection limit of the GES-LAMP method was assessed using purified DNA and DNA-spiked clinical samples (urine, sputum, and blood). To determine the clinical usefulness of the methods, we used different (genotypically and phenotypically) P. aeruginosa clinical isolates, collected from diverse geographical locations between 2003 and 2012. The novel LAMP assay targeting blaGES was highly specific. The detection limit was 10 DNA copies per reaction; the assay was 10-fold more sensitive than conventional PCR. The LAMP assay detected blaGES with high sensitivity in all DNA-spiked samples; PCR did not detect blaGES in blood samples. The GES-LAMP method correctly detected the 5 isolates containing blaGES among the 14 isolates tested. Using these isolates, we confirmed that our Carba-GES-LAMP method of detecting point mutations correctly identified the two blaGES positive organisms with carbapenemase activity. To the best of our knowledge, this is the first report of the GES β-lactamase gene detection assay using the LAMP method. Our new assays effectively detect blaGES and critical unique mutations.

Classification of Multiple DNA Dyes Based on Inhibition Effects on Real-Time Loop-Mediated Isothermal Amplification (LAMP): Prospect for Point of Care Setting

“…Real-time LAMP has been employed since it is not only effective for DNA amplification under isothermal conditions but also simultaneously offers detection, which can be quantitative for e.g., the monitoring of specific gene expression (Mori et al, 2004; Cai et al, 2008; Chen and Ge, 2010; Tourlousse et al, 2012; Cao et al, 2015) or for detection of bacterial concentrations (Nixon et al, 2014). Measurement of turbidity has been used in real-time LAMP (Takano et al, 2019) and a commercial detection device is available (Eiken Chemical, Co., Ltd., Japan). The real-time LAMP turbidity is based on quantification of an increase of magnesium pyrophosphate precipitate as a by-product of the LAMP reaction (Mori et al, 2004).…”

Portable real-time colorimetric LAMP-device for rapid quantitative detection of nucleic acids in crude samples

“…The latter is important not only for lab-based but also for point-of-care applications as, for example, in the case of HIV viral load monitoring in resourcelimited areas 14,17 . However, the bulky and not so sensitive turbidimeters 18,19 and expensive realtime fluorescence-machines are not ideal for POC diagnostics. The same applies to the use of sophisticated but costly microfluidic chips 20,21 often used for real time monitoring in combination with optical 22 , electrochemical 23 , smartphone [24][25][26] , acoustic 27 or other microdevices 22,28,29 .…”

Image-guided study of swine anatomy as a tool for urologic surgery research and training

“…Suprapubic access with catheter placement is an alternative used for this model 58 . Large White, Landrace, Yorkshire and Duroc females, weighing 30–40 kg, were successfully submitted to ureteral catheterization, although difficulty was reported during the procedure 59 , 60 .…”