Rapid detection of a novel B1-β-lactamase gene, blaAFM-1 using a loop-mediated isothermal amplification (LAMP) assay

Abstract

Background BlaAFM-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. Methods We 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. Results The 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. Conclusions We established a new LAMP assay with high sensitivity and specificity to detect the novel B1-β-lactamase gene, blaAFM-1.

Rapid on-site nucleic acid testing: On-chip sample preparation, amplification, and detection, and their integration into all-in-one systems

“…LAMP uses six regions for initial recognition of the target sequence, and then uses four primers during the subsequent amplification, elongation, and recycling processes; this provides very high specificity. In some studies, two additional primers have been used to further increase specificity ( Qin Y et al, 2021 ). Unlike PCR, which produces exact duplicates of the template DNA, the LAMP reaction produces dumbbell-shaped or stem-loop DNA structures with different lengths.…”

Characterization of blaAFM-1-positive carbapenem-resistant strains isolated in Guangzhou, China

“…All strains were recovered from the feces of the inpatient patients. AN70, NFYY023, E202, and NCTC10498 strains were verified as carrying bla AFM-1 gene in the previous study [ 10 ]. AN70, NFYY023, E202, and NCTC10498 strains were grown on plates containing 4 μg/ml meropenem and incubated at 35 °C for 18 h. Antimicrobial susceptibility testing (AST) of isolates was performed by broth microdilution assay and the minimal inhibitory concentration (MIC) values were interpreted by Clinical And Laboratory Standard Institute (CLSI) document M100-32ed [ 15 ].…”

“…Using pET-28a( +) plasmid and E. coli Top10 competent cells as plasmid vector and expression vector to generate AFM-1 clones (pET-28a( +)-AFM-1-Top10). The specific operation is the same as the previously described [ 10 ]. Positive clones were screened with 4 μg/ml meropenem.…”

“…Bla AFM-1 (AFM standing for Alcaligenes faecalis metallo ), was first found in Alcaligenes faecalis strain AN70 ( A. faecalis AN70), and later several Gram-negative strains such as Comamonas testosteroni NFYY023 ( C. testosteroni NFYY023), Bordetella trematum E202 ( B. trematum E202), and Stenotrophomonas maltophilia NCTC10498 ( S. maltophilia NCTC10498) that carried bla AFM-1 gene were screened out by our LAMP assay [ 10 ]. Next, other researchers found that the bla AFM-1 gene was also identified on the plasmids of an Aeromonas hydrophila ( A. hydrophila ) isolate [ 11 ] and Pseudomonas aeruginosa ( P. aeruginosa ) isolates [ 12 ].…”

“…In this study, we reported a separate B1 carbapenemase, designated to NCBI on 16 November 2018 to get an accession number, we named it AFM-1, which is an acronym for ‘ Alcaligenes faecalis metallo enzyme’ and exhibited 86% amino acid identity to NDM-1. We screened four species carrying the bla AFM-1 gene by the LAMP detection method, as previously reported [ 10 ]. Intriguingly, some researchers have also found this gene carried by other species [ 11 , 12 ].…”