Detection of Carbapenemases by Real-Time PCR and Melt Curve Analysis on the BD Max System

Abstract

bWe developed a multiplex SYBR green real-time PCR for the BD Max instrument (BD Diagnostics, Sparks, MD) to detect a panel of carbapenemases. The assay was evaluated with 152 consecutive isolates sent to the German National Reference Laboratory, and 65/65 of the carbapenemase-positive and 87/87 of the carbapenemase-negative strains were identified correctly. R apid detection and differentiation of carbapenemases are important for epidemiological investigations and infection control measures, thereby contributing to the prevention of global spread and optimization of antimicrobial therapy (1). Phenotypebased techniques can be used to identify carbapenemases (2-7), yet the specificity and sensitivity of those procedures vary. Other assays use matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF) (8), enzymatic measurements like the Carba NP test (9), and spectrophotometry (10). However, phenotypic and functional assays are often followed by molecular detection of the encoding carbapenemase genes. Molecular analysis is laborious, but fully automated PCR platforms might circumvent this restriction. Multiplexing allows detection in an economically reasonable manner (11).In our study, SYBR green real-time PCR on the BD Max system (BD Diagnostics, Sparks, MD) combined with melt curve analysis was developed to detect frequent carbapenemases in cultured isolates. Primers were mixed in two multiplex PCRs (master mix 1 [MM1] and MM2) that were selected on differences in the melting points of the amplicons. Panel 1 (MM1) amplified IMP-1, IMP-2, GES, KPC, VIM-2, and 16S rRNA as an internal amplification control. Panel 2 (MM2) detected OXA-23-like, VIM-1, OXA-48-like, and NDM (see Table S1 in the supplemental material). One to 2 colonies of a suspect isolate were added to 0.5 ml of 10 mM Tris-1 mM EDTA (pH 8.0) containing 0.1-mm glass beads (BioSpec, Bartlesville, OK). The sample was vortexed and incubated at 95°C for 10 min, and the cleared supernatant was directly used for the PCR. Each single reaction consisted of forward and reverse primers for each of the carbapenemases (final concentration, 0.25 M), a primer for 16S rRNA (only MM1, 0.05 M), 6.25 l of 2ϫ ABsolute SYBR green mix No Rox (Thermo Scientific, Schwerte, Germany), and PCR-grade water up to 10 l. Then 2.5 l of the strain lysate was combined with 10 l of the respective master mix and pipetted manually into a microfluidic BD Max PCR cartridge. The PCR was run in the PCR-only mode at 95°C for 15 min for 30 PCR cycles (98°C, 30 s; 56°C, 20 s; 72°C, 51.1 s) followed by melt curve analysis from 60°C to 100°C in 0.2°C steps.Using plasmids encoding the various carbapenemases, we observed clearly distinct melting temperatures ( Fig. 1A and B). Only IMP-1 and IMP-2 could not be differentiated. Melting temperatures were consistent and highly reproducible (Fig. 1C). The strains harboring a carbapenemase gene could be identified by comparison to the reference temperatures obtained with plasmids (an example is shown in Fig. 1D). Even the co...

Rapidec Carba NP Test for Rapid Detection of Carbapenemase Producers

“…Few techniques are available for the rapid identification of carbapenemase producers (4) and include UV spectrophotometry (5), matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) technology (6)(7)(8), and molecular techniques (9)(10)(11)(12). These techniques have overall good sensitivities and specificities but may require trained microbiologists, expensive equipment, and may be time-consuming and expensive.…”

Rapid Detection of KPC, NDM, and OXA-48-Like Carbapenemases by Real-Time PCR from Rectal Swab Surveillance Samples

“…Although there have been several reports of real-time PCR assays in the literature (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14), to our knowledge, this is the first Fast multiplex real-time 5= exonuclease-based real-time PCR to detect and differentiate the 3 carbapenemases, KPC, NDM, and OXA-48-like, from rectal swabs in enrichment broths in a single reaction. A culture enrichment step was incorporated to increase the sensitivity (15 PCR controls, ATCC strains, and clinical isolates previously characterized by conventional PCR (16) are listed in Table 2.…”

“…Molecular methods are fast and generally have higher sensitivity and specificity than phenotypic methods, and methods developed in-house are typically less expensive than commercially available kits. The published methods for the molecular detection of Klebsiella pneumoniae carbapenemases (KPC), New Delhi metallo-␤-lactamases (NDM), and OXA-48 are typically singleplex reactions targeting only one carbapenemase or are intended for cultured isolates rather than surveillance samples (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14). We identified the need for a highly sensitive and specific molecular assay that can detect these CPO gene targets directly from surveillance sample matrices, such as rectal swabs.…”

Development of a Real-Time PCR Protocol Requiring Minimal Handling for Detection of Vancomycin-Resistant Enterococci with the Fully Automated BD Max System

“…A recent new device for fully automated molecular detection is the BD Max system, which combines nucleic acid extraction, PCR setup, and 5-color real-time PCR detection. The flexible programming allows user-developed assays to be run in an automated manner (21)(22)(23)(24)(25). However, those assays still require liquid handling and thus need a laboratory capable of performing molecular assays.…”