Multiplex Real-Time PCR Assay for Six Major Carbapenemase Genes

Abstract

The global dissemination of carbapenemase-producing Enterobacteriaceae (CPE) is a major concern in public health. Due to the existence of the diversity of carbapenemases, development of an easily available, cost-effective multiplex detection assay for CPE is required worldwide. Using clinically available and reliable equipment, COBAS® z480 (Roche Diagnostics K.K., Tokyo, Japan), we developed a multiplex real-time PCR assay for the detection of two combinations of carbapenemases; first, blaNDM, blaKPC, and blaIMP (Set 1), and second, blaGES, blaOXA-48, and blaVIM (Set 2). We constructed standard curves for each carbapenemase gene using serial dilutions of DNA standards, then applied reference or clinical isolates with each carbapenemase gene to this assay. The multiplex assay showed satisfactory accuracy to detect CPE genes, with the correlation coefficients of greater than 0.99 for all genotypes. The assay appropriately differentiated the reference or clinical strains harboring each carbapenemase gene without cross reactivity. Lastly, the assay successfully detected multiple genes without false-positive reactions by applying six clinical isolates carrying both NDM and OXA-48-like carbapenemase genes. Major advantages of our assay include multiplicity, simple operation, robustness, and speed (1 h). We believe that the multiplex assay potentially contributes to early diagnosis of CPE with a diverse genetic background.

Clinical and Molecular Profile of Carbapenem Resistant Klebsiella pneumoniae Infections in a Tertiary Care Hospital –Mangalore

“…This technique is used to amplify the targeted DNA sequence by use of hydrolysis probes that are short oligonucleotides, that have a fluorescent reporter dye attached to the 5’end and a quencher dye to the 3’end. The details of primers and probes 22 for blaNDM, blaKPC, blaIMP, blaOXA-48, and blaVIM are shown in Table 1 . We used two multiplex assays to identify two combinations of five carbapenemase genes and an internal control; Set 1 and Set 2 contained blaNDM, blaKPC, blaIMP, blaVIM, and blaOXA-48, respectively.…”

“…In a single-tube reaction, the kit enables the accurate and precise detection of both single and co-present carbapenemase-encoding genes. 22 , 23 …”

Carbapenemase-Producing Klebsiella pneumoniae Infections in Diabetic and Nondiabetic Hospitalized Patients

“…Next, two wells were filled with 10 µL of inoculum, one of which contained imipenem. Gram-negative bacteria that produced metallo β lactamases (MBL) were treated with imipenem as the carbapenemase zinc substrate; positive results were defined as those in which the color of the bacteria changed from red to yellow, orange, or thick orange in comparison to the control well [17][18][19].…”

“…Cycle threshold values greater than ≤40 were regarded as positive. The kit enables the accurate and precise detection of carbapenemase encoding genes that are present singly or in combination in single tube reactions [19,20].…”

Carbapenem resistance determinants in Klebsiella pneumoniae strains isolated from blood cultures-comparative analysis of molecular and phenotypic methods

“…Yoshioka et al used two combinations of carbapenemases detection primers, as we did, but selected partially different primers. They concluded that the assay appropriately differentiated the reference or clinical strains harboring each carbapenemase gene without cross reactivity(25). LightMix® Modular carbapenemases Kits detect the main classes of carbapenemases with a high level of accuracy.…”