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Comparative Study
. 2012 May;50(5):1580-5.
doi: 10.1128/JCM.06647-11. Epub 2012 Feb 22.

Sensitive and Rapid Detection of the New Delhi Metallo-Beta-Lactamase Gene by Loop-Mediated Isothermal Amplification

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Free PMC article
Comparative Study

Sensitive and Rapid Detection of the New Delhi Metallo-Beta-Lactamase Gene by Loop-Mediated Isothermal Amplification

Wei Liu et al. J Clin Microbiol. .
Free PMC article

Abstract

New Delhi metallo-β-lactamase 1 (NDM-1), which is associated with resistance to carbapenem, was first reported in 2008. A sensitive and rapid molecular assay to detect the plasmid bla(NDM-1) in clinical isolates is needed to control its spread. We describe a loop-mediated isothermal amplification (LAMP) assay for the rapid detection of bla(NDM-1) from pure culture and sputum, urine, and fecal samples. Eight sets of primers were designed to recognize six or eight distinct sequences on target bla(NDM-1), and one set was selected as the most appropriate set of primers for its rapid detection. The specificity and sensitivity of the primers in the LAMP reactions for bla(NDM-1) detection were determined. The sensitivity of the LAMP assay for bla(NDM-1) detection in sputum, urine, and fecal samples was also tested. Two methods, namely, monitoring of turbidity and addition of calcein to the reaction tube, were used to determine negative and positive results. The results showed that target DNA was amplified and visualized by the two detection methods within 70 min at an isothermal temperature of 65°C. The sensitivity of LAMP, with a detection limit of 10.70 pg/μl DNA, was 100-fold greater than that of PCR. Thirteen infection bacterial strains without bla(NDM-1) were selected for testing of specificity, and the results of the amplification were negative, which showed that the primers had good levels of specificity. The LAMP method reported here is demonstrated to be a potentially valuable means for the detection of bla(NDM-1) and rapid clinical diagnosis, being fast, simple, and low in cost.

Figures

Fig 1
Fig 1
Eight sets of primers amplified the target gene under the same conditions. Turbidity was monitored by a Loopamp real-time turbidimeter at 400 nm every 6 s. Assays with the CJXJ1 and CJXJ2 primer sets were performed with Loop primers; assays with the other sets were performed without Loop primers.
Fig 2
Fig 2
Different temperatures of the LAMP reaction for detection of NDM-1. Turbidity was monitored by a Loopamp real-time turbidimeter at 400 nm every 6 s.
Fig 3
Fig 3
Specificity of the LAMP reaction for detection of blaNDM-1. Turbidity was monitored by a Loopamp real-time turbidimeter at 400 nm every 6 s. Amplification was performed at 65°C for 65 min. Lines: 1, negative control (double-distilled water); 2, A. baumannii XM; 3, A. baumannii H949; 4, A. baumannii F398; 5, A. baumannii B260; 6, A. baumannii H18; 7, S. sonnei 2531; 8, S. flexneri 4536; 9, S. enterica serotype Enteritidis 50326-1; 10, V. carchariae 5732; 11, S. enterica serotype Paratyphi 86423; 12, enteroinvasive E. coli 44825; 13, enterotoxigenic E. coli 44824; 14, enteropathogenic E. coli 2348; and 15, V. parahaemolyticus 5474.
Fig 4
Fig 4
Comparison of sensitivity between the LAMP reaction and PCR for detection of the blaNDM-1 gene. The pure genomic DNA extracted from A. baumannii XM was diluted in a serial 10-fold dilution. Both LAMP reactions (A and B) and PCRs (C) were carried out in duplicate for each dilution point. Tubes and lanes: 1, 1,070 ng/μl; 2, 107.0 ng/μl; 3, 10.70 ng/μl; 4, 1.070 ng/μl; 5, 107.0 pg/μl; 6, 10.70 pg/μl; 7, 1.070 pg/μl; 8, 0.107 pg/μl. (A) Turbidity was monitored by a Loopamp real-time turbidimeter at 400 nm every 6 s; (B) 1 μl of fluorescent detection reagent was added to 25 μl of LAMP reaction mixture before the LAMP reaction; (C) the PCR products were analyzed by 2% agarose gel electrophoresis and stained with ethidium bromide.
Fig 5
Fig 5
Detection of the blaNDM-1 gene in simulated sputum samples (A), simulated urine samples (B), and simulated fecal samples (C) by a Loopamp real-time turbidimeter at 400 nm every 6 s. The concentration of pure genomic DNA extracted from A. baumannii XM in each simulated sputum sample is shown.

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