Multiplex High-Resolution Melting Analysis as a Diagnostic Tool for Detection of Plasmid-Mediated AmpC β-Lactamase Genes

Abstract

High-resolution melting (HRM) analysis can be a diagnostic tool to evaluate the presence of resistance genes with the added bonus of discriminating sequence modifications. A real-time, multiplex PCR assay using HRM was designed for the detection of plasmid-mediated ampC genes. The specificity and sensitivity of this assay were 96% and 100%, respectively. P lasmid-mediated AmpC ␤-lactamase (pmAmpC) genes are derived from the chromosomal ampC genes from various Gram-negative bacterial species. These genes are divided into six families, including bla MOX , bla FOX , bla CMY-2 , bla DHA , bla ACC , and bla ACT . AmpC ␤-lactamases are associated with resistance to broad-spectrum penicillins, broad-spectrum cephalosporins, monobactams, and cephamycins (1-4). The establishment of standardized procedures for the identification of pmAmpC-producing organisms has yet to occur, thereby allowing this resistance mechanism to spread throughout community and hospital settings or be misidentified as an extended-spectrum ␤-lactamase (1, 2). Therapeutic failure has been associated with the inability to detect a resistant phenotype when the organism produced a pmAmpC (5, 6). AmpC multiplex PCR assays previously designed by our laboratory have been modified into a realtime, multiplex assay that uses high-resolution melting (HRM) analysis to detect all six families of pmAmpC genes in Ͻ2 h (7,8). Using this technology, each ampC gene fragment produces an amplicon with distinct melt characteristics to yield six separated melt curves. Primers used in the assay are listed in Table 1 and annealed with 100% specificity to target gene variants (7,8).DNA was extracted from an overnight culture using the FDAapproved QIAamp DSP DNA minikit (Qiagen, Hilden, Germany). The final reaction mixture volume for the multiplex PCR was 25 l. Each PCR mixture consisted of 1ϫ EpiTect Type-iT HRM PCR buffer and the following primer concentrations: 0.6One microliter of DNA template (10 ng/l) was added to 24 l of the master mix. PCR parameters included an initial denaturation at 95°C for 5 min, followed by three-step cycling conditions for 40 cycles consisting of denaturation at 95°C for 10 s, annealing at 55°C for 30 s, and extension at 72°C for 10 s. Fluorescence acquisition was programmed to the green channel on the extension step of PCR amplification. When using the Rotor-Gene Q, only 10 reactions could be prepared in one master mix without observing a flat line of fluorescence, and therefore, a minimum of two master mixes had to be prepared for each real-time PCR run. HRM analysis followed the cycling parameters with a temperature range of 80 to 89°C that was increased in 0.1°C increments. ScreenClust analysis was attempted to simplify interpretation of HRM results but was unsuccessful in generating the appropriate clusters (Qiagen, Hilden, Germany) (9-11).All primer sets were evaluated for their ability to anneal to target genes using a previously constructed panel of K12 transformants containing pmAmpC gene fragments and HRM analysis using the conditio...

New approach to identify colistin‐resistant Pseudomonas aeruginosa by high‐resolution melting curve analysis assay

“…High-resolution melting (HRM) analysis is a recently developed technique for fast, high-throughput post-PCR analysis of genetic mutations or variance in nucleic acid sequences. (Geyer and Hanson 2014). In HRM, the fluorescence produced by a DNA intercalating dye (e.g.…”

A High Resolution DNA Melting Curve Analysis for the Rapid and Efficient Molecular Diagnostics of Extended Spectrum β-Lactamase Determinants from Foodborne Escherichia coli

“…HRM assays have also been previously developed for the typing and subtyping of both microbial species and β-lactamases, most frequently isolated from certain hospital settings. For instance, HRM protocols were developed and tested for detection of TEM, SHV, and CTX-M beta-lactamases in E. coli, Klebsiella pneumoniae, and Enterobacter cloacae from patients admitted in hospitals [21,22] and for detection and identification of metallo-β-lactamases [24]. Chia et al [22] developed an efficient multiplex PCR targeting bla(SHV), bla(CTX-M-3)-like, and bla(CTX-M-14)-like genes and a modified SHV melting-curve mutation detection method to rapidly distinguish six prevalent bla(SHV) genes (bla(SHV-1), bla(SHV-2), bla(SHV-2a), bla(SHV-5), bla(SHV-11), and bla(SHV-12)) in Taiwan hospitals.…”

“…HRM methods have been developed and applied for the identification and subtyping of bacterial and viral pathogens [19,21], genes encoding selected β-lactamases from hospital settings [22,23], AmpC β-lactamases [24], Metallo-β-Lactamase [25], and multidrug-resistant Mycobacterium tuberculosis [20] mostly of clinical microbiology concern. The most prevalent ESBLs and their variants associated with foods and food animals [15] differ from those targeted by HRM protocols developed for isolates from clinical settings.…”

A highly multiplexed melt-curve assay for detecting the most prevalent carbapenemase, ESBL and AmpC genes

“…This technique has a greater potential for multiplexing than probe based assays, as it is not limited by the number of channels available in the qPCR system 31 . HRM assays have been designed for bacterial resistance genes, including carbapenemases 32–34 , AmpCs 35 , OXA-48-like beta-lactamases 36 , chloramphenicol resistance 37 and mutations in aac(6’)-lb-cr 38 . However, current assays include a maximum of six targets, and multiple tests are required to thoroughly detect resistance to a drug type.…”