Preliminary evaluation of a multiplex RT-PCR kit in development for detecting bacterial resistance genes to carbapenems

Introduction: Antibiotic bacterial resistance is a natural biological phenomenon.However, their use in the community and hospital environment are risk factors for the selection and spread of bacterial resistance.Emerged multidrug-resistant bacteria can produce an enzyme called carbapenemase, as one of the mechanisms of resistance to antibiotics.Which represents high treatment costs and high mortality rates.KPC, NDM, VIM and IMP are the most found carbapenemases in bacteria, with very rapid worldwide dissemination.Objectives: Develop a commercial multiplex kit to detect carbapenemase (KPC, NDM, VIM, IMP) and internal control in patients with symptoms.Methodology: Strains from the Unified Health Surveillance Culture Collection (CCVSU) were donated by the National Institute for Quality Control in Health (INCQS/Fiocruz-RJ).A matrix of combinations between primers and probes (FAM, RED and Cy5) of the KPC and NDM targets were suggested.Six mixes were produced with concentrations of 0.2 uM, 0.2uM and 0.15 uM for sense, antisense primers and probe of the KPC and 16rRNA targets and 0.4uM, 0.4uM and 0.2uM for NDM.The samples were tested in duplicate.The endogenous control used was the constitutive gene 16SrRNA.The reaction occurred with colony lysis directly in the real-time PCR reaction (PCR colony).The cycling used was 95 C for 10 minutes (initial denaturation) and 45 cycles of 95 C for 5 seconds and 60 C for 30 seconds.The results were analyzed using the Design & Analysis 2.8.0 software. Results:Until now, a triplex assay for blaKPC, blaNDM-1 and 16SrRNA has been performed with satisfactory performance for three of the six mixes tested: (i) KPC FAM and NDM Cy5; (ii) KPC Cy5 and NDM FAM and (iii) KPC Cy5 and NDM RED, the internal control with NED probe.Among them, the KPC Cy5 and NDM FAM mix presented the best Ct averages: KPC Ct 16.8 and NDM Ct 35.3. Conclusion:The preliminary results suggest that the development of a multiplex kit for detecting the main carbapenemases KPC, NDM, VIM and IMP have a promising future.And it could serve as a surveillance mechanism for the presence and possible dissemination of these resistant microorganisms in the hospital environment.