Rapid and accurate detection of carbapenem-resistance gene by isothermal amplification in Acinetobacter baumannii

Abstract

Background Acinetobacter baumannii (A. baumannii) is one of the pivotal pathogens responsible for nosocomial infections, especially in patients with low immune response, and infection with carbapenem-resistant A. baumannii has been increasing in recent years. Rapid and accurate detection of carbapenem-resistance genes in A. baumannii could be of immense help to clinical staff. Methods In this study, a 15-μL reaction system for recombinase polymerase amplification (RPA) was developed and tested. We collected 30 clinical isolates of A. baumannii from the Burn Institute of Southwest Hospital of Third Military Medical University (Army Medical University) for 6 months and tested antibiotic susceptibility using the VITEK 2 system. A. baumannii was detected based on the blaOXA-51 gene by PCR, qPCR and 15 μL-RPA, respectively. Sensitivity and specificity were evaluated. In addition, PCR and 15 μL-RPA data for detecting the carbapenem-resistance gene blaOXA-23 were comparatively assessed. Results The detection limit of the blaOXA-51 gene by 15 μL RPA was 2.86 CFU/ml, with sensitivity comparable to PCR and qPCR. No positive amplification signals were detected in non-Acinetobacter isolates, indicating high specificity. However, only 18 minutes were needed for the 15 μL RPA assay. Furthermore, an antibiotic susceptibility test showed that up to 90% of A. baumannii strains were resistant to meropenem and imipenem; 15 μL RPA data for detecting blaOXA-23 showed that only 10% (n = 3) of A. baumannii isolates did not show positive amplification signals, and the other 90% of (n = 27) isolates were positive, corroborating PCR results. Conclusion We demonstrated that the new 15 μL RPA assay for detecting blaOXA-23 in A. baumannii is faster and simpler than qPCR and PCR. It is a promising alternative molecular diagnostic tool for rapid and effective detection of A. baumannii and drug-resistance genes in the field and point-of-care testing.

Recent advances in recombinase polymerase amplification: Principle, advantages, disadvantages and applications

“…established a 15 µl RPA reaction system to detect carbapenem-resistance genes blaoxa-23 of Acinetobacter baumannii. The test results showed that 90% of the strains showed positive amplification signals, and only 10% of the strains showed negative amplification signals, which was consistent with the results of the PCR ( Liu et al., 2020 ). Methicillin-resistant staphylococci (MRS) was a common drug-resistant strain in the clinic.…”

Development and Clinical Application of a Recombinase Polymerase Amplification-Lateral Flow Strip Assay for Detection of Carbapenem-Resistant Acinetobacter baumannii

“…baumannii and 10 1 CFU for CRAB. This sensitivity was the same as that of the real-time RPA method, which was in the range of 10 0 –10 1 CFU per reaction ( Liu et al., 2020 ). In addition, the RPA-LFS assay for detection of CRAB was simple and fast, as detection can be completed within 40 min (30 min for amplification and 10 min for LFS analysis).…”

A Rapid Antimicrobial Resistance Diagnostic Platform for Staphylococcus aureus Using Recombinase Polymerase Amplification

“…During the recent period, several studies have proven the exceptionally high capability of RPA for selectively recognizing microorganisms and genes of interest. This capacity has exposed RPA as a valuable POCT technique in clinical microbiology research ( 35 – 40 ). Several writers have previously proven the possibility of RPA or RPA incorporating various aptamer technologies, such as multicomponent nucleic acid enzymes (MNAzymes)-based techniques and CRISPR/Cas-based techniques ( 41 , 42 ), for the identification of AMR S. aureus strains.…”