title: Sensitive and rapid detection of the new Delhi metallo-beta-lactamase gene by loop-mediated isothermal amplification.
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.
authors.affiliation: Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Liu
authors.forename: Dayang
authors.initials: D
authors.lastname: Zou
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
authors.forename: Xuesong
authors.initials: X
authors.lastname: Wang
authors.forename: Xiang
authors.initials: X
authors.lastname: He
authors.forename: Xiao
authors.initials: X
authors.lastname: Wei
authors.forename: Changlin
authors.initials: C
authors.lastname: Shao
authors.forename: Xuelian
authors.initials: X
authors.lastname: Li
authors.forename: Wei
authors.initials: W
authors.lastname: Shang
authors.forename: Kaitao
authors.initials: K
authors.lastname: Yu
authors.forename: Dawei
authors.initials: D
authors.lastname: Liu
authors.forename: Yunmei
authors.initials: Y
authors.lastname: Li
authors.forename: Jing
authors.initials: J
authors.lastname: Guo
authors.forename: Zhitao
authors.initials: Z
authors.lastname: Yin
authors.forename: Jing
authors.initials: J
authors.lastname: Yuan
mesh_terms: D001419:Bacteria / Q000201:enzymology* / Q000235:genetics; D001431:Bacteriological Techniques / Q000379:methods*; D017931:DNA Primers / Q000235:genetics; D005243:Feces / Q000382:microbiology; D006801:Humans; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D010957:Plasmids; D012680:Sensitivity and Specificity; D013183:Sputum / Q000382:microbiology; D013696:Temperature; D013997:Time Factors; D014556:Urine / Q000382:microbiology; D001618:beta-Lactamases / Q000235:genetics*
chemical_list: D017931:DNA Primers; D001618:beta-Lactamases; C552864:beta-lactamase NDM-1