Rapid Detection of the Blatem Gene in Acinetobacter Baumannii Using Rpa Isothermal Amplification and the Crispr/Cas12a System

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.