Development Of A Rapid Molecular Test Format Based On Isothermal Recombinase Polymerase Amplification Technique And Lateral Flow Method For Detection Of Bla(Oxa-48)

Objective: Molecular tests are rapid, reliable tools for the detection of carbapenem resistance, but their use is limited due to their cost, requirement for well-trained technicians and highly sophisticated instrumentation. The recombinase polymerase amplification (RPA) assay, one of the isothermal amplification methods developed recently to overcome these problems, doesn't require denaturation of target, and RPA products can be determined by probe-base detection methods even without a specific instrumentation. In this study, we aimed to develop a rapid and easily applicable molecular test format in on-site settings based on RPA technique with combination of lateral flow system for detection of bla(OXA-48).Methods: Bacterial strains were obtained from the collection of hospital infection control committee. Twenty-four OXA-48-, one from each of VIM-, IMP-, NDM-, KPC-positive strains and 10 carbapenem-susceptible OXA-48-negative strains were included. Fresh cultures were suspended in 1x polymerase chain reaction (PCR) buffer, the turbidity of the suspensions were adjusted to 0.5 McFarland standard. Serial dilutions of OXA48- positive strain were prepared for evaluation of test performance and detection of lowest limit. Nucleic acid isolation was performed by boiling method. bla(OXA-48)-specific RPA primers and NFO probe were designed and used. RPA reactions were performed according to the manufacturer's (TwistAmp (R) nfo kit, TwistDx, Cambridge, United Kingdom) instructions. Lateral flow method was used for detection of RPA products.Results: OXA-48-positive strains were detected within about 45 minutes without any need for special instruments. Lowest detection limit of the test was 10 bacteria. Neither cross-reactions nor false positivity was observed with the strains having other resistance genes or carbapenem-susceptible strains.Conclusions: Based on its simplicity and applicability without any need for special instruments, the newly-developed test can be an alternative tool to the other molecular tests (like PCR or real-time PCR) for screening of these type of enzymes for both laboratory and on-site settings.