RAPID DETECTION OF THE BLATEM GENE IN ACINETOBACTER BAUMANNII USING RPA ISOTHERMAL AMPLIFICATION AND THE CRISPR/CAS12A SYSTEM
Main Article Content
Authors
M. Amanzholova
National Center for Biotechnology, 13/5 Kurgalzhynskoye road, Astana, 010000, Kazakhstan
Farabi Kazakh National University, 71 Al-Farabi Avenue, Almaty, 050040, Kazakhstan
A. Akimbekova
National Center for Biotechnology, 13/5 Kurgalzhynskoye road, Astana, 010000, Kazakhstan
A. Shaizadinova
National Center for Biotechnology, 13/5 Kurgalzhynskoye road, Astana, 010000, Kazakhstan
N. Sutimbekova
Astana Medical University, 33 Saryarka Avenue, Astana, 010000, Kazakhstan
N. Bissenova
National Scientific Medical Center, 42 Abylai Khan Avenue, Astana, 010000, Kazakhstan
P. Tarlykov
National Center for Biotechnology, 13/5 Kurgalzhynskoye road, Astana, 010000, Kazakhstan
S. Abeldenov
National Center for Biotechnology, 13/5 Kurgalzhynskoye road, Astana, 010000, Kazakhstan
Abstract
Antibiotic resistance is one of the key problems of modern medicine, especially in relation to nosocomial infections caused by Acinetobacter baumannii, which has a high ability to accumulate drug resistance genes. One of the beta-lactam antibiotic resistance genes is blaTEM, which plays an important role in the formation of resistance in gram-negative microorganisms, including Acinetobacter baumannii. Standard molecular genetic methods for detecting antibiotic resistance genes, including PCR and real-time PCR, require the use of thermal cycling equipment. In this regard, the development of isothermal amplification methods with alternative methods of recording the result is of interest to laboratories with limited instrumentation. In this regard, the use of isothermal amplification methods in combination with CRISPR/Cas systems for sensitive detection of target genes is a promising direction. The purpose of this study was to develop and evaluate the sensitivity of a method for detecting the blaTEM gene based on recombinase polymerase amplification (RPA) in combination with the CRISPR/Cas12a system. The study showed that the RPA method provides specific amplification of the target gene with a detection limit of 104 copies/reaction. The results obtained confirm the fundamental operability of the RPA-CRISPR/Cas12a system for detecting a fragment of the blaTEM gene and fluorescence registration of the amplification product.
Keywords
Acinetobacter baumannii, blaTEM, antibiotic resistance, RPA, CRISPR/Cas12a, isothermal amplification, Molecular diagnostics
Article Details
References
Sharma, R. and D. Lakhanpal, Acinetobacter baumannii: A comprehensive review of global epidemiology, clinical implications, host interactions, mechanisms of antimicrobial resistance and mitigation strategies. Microb Pathog, 2025. 204: p. 107605.
Global burden of bacterial antimicrobial resistance 1990-2021: a systematic analysis with forecasts to 2050. Lancet, 2024. 404(10459): p. 1199-1226.
Aggarwal, R., et al., Antibiotic resistance: a global crisis, problems and solutions. Crit Rev Microbiol, 2024. 50(5): p. 896-921.
Bergogne-Bérézin, E. and K.J. Towner, Acinetobacter spp. as nosocomial pathogens: microbiological, clinical, and epidemiological features. Clin Microbiol Rev, 1996. 9(2): p. 148-65.
Kyriakidis, I., et al., Acinetobacter baumannii Antibiotic Resistance Mechanisms. Pathogens, 2021. 10(3).
Wang, M., et al., Clinical Outcomes and Bacterial Characteristics of Carbapenem-resistant Acinetobacter baumannii Among Patients From Different Global Regions. Clin Infect Dis, 2024. 78(2): p. 248-258.
Castañeda-Barba, S., E.M. Top, and T. Stalder, Plasmids, a molecular cornerstone of antimicrobial resistance in the One Health era. Nat Rev Microbiol, 2024. 22(1): p. 18-32.
Ranjbar, R. and A. Farahani, Study of genetic diversity, biofilm formation, and detection of Carbapenemase, MBL, ESBL, and tetracycline resistance genes in multidrug-resistant Acinetobacter baumannii isolated from burn wound infections in Iran. Antimicrob Resist Infect Control, 2019. 8: p. 172.
Davin-Regli, A., J.-P. Lavigne, and J.-M. Pagès, Enterobacter spp.: Update on Taxonomy, Clinical Aspects, and Emerging Antimicrobial Resistance. Clinical Microbiology Reviews, 2019. 32(4): p. 10.1128/cmr.00002-19.
Sinha, S. and L.S.B. Upadhyay, Understanding antimicrobial resistance (AMR) mechanisms and advancements in AMR diagnostics. Diagn Microbiol Infect Dis, 2025. 113(2): p. 116949.
Yan, W.X., et al., Functionally diverse type V CRISPR-Cas systems. Science, 2019. 363(6422): p. 88-91.
Li, S.Y., et al., CRISPR-Cas12a has both cis- and trans-cleavage activities on single-stranded DNA. Cell Res, 2018. 28(4): p. 491-493.
Akimbekova, A., et al., Integrated RPA-CRISPR/Cas12a Technology for Rapid Detection of Salmonella enterica. Diagnostics (Basel), 2026. 16(9).
Zhang, X., et al., Rapid and Specific Detection of Acinetobacter baumannii Infections Using a Recombinase Polymerase Amplification/Cas12a-based System. J Vis Exp, 2025(218).
Zhao, Z., et al., Development of a CRISPR/Cas-Based Detection Platform for Tracking Decreased Susceptibility to Cephalosporins in Neisseria gonorrheae. Anal Chem, 2025. 97(36): p. 19445-19455.
Shin, J., et al., A CRISPR/Cas12a-Based System for Sensitive Detection of Antimicrobial-Resistant Genes in Carbapenem-Resistant Enterobacterales. Biosensors (Basel), 2024. 14(4).