Rapid and Specific Detection of Acinetobacter baumannii Infections Using a Recombinase Polymerase Amplification/Cas12a-based System

Xia Zhang; Meina Duan; Yuzhe Zhao; Kaifeng Zhang; Fei Liu; Jing Jie; Chunxiuli Li; Dong Chen; Dan Li; Shucheng Hua; Chunyan Wang; Qingtian Guan; Jing Wu; Bing Liu; Lei Song

doi:10.3791/67542

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Method Article

Rapid and Specific Detection of Acinetobacter baumannii Infections Using a Recombinase Polymerase Amplification/Cas12a-based System

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DOI:

10.3791/67542

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April 25th, 2025

Xia Zhang¹ , Meina Duan² , Yuzhe Zhao² , Kaifeng Zhang² , Fei Liu² , Jing Jie² , Chunxiuli Li² , Dong Chen² , Dan Li² , Shucheng Hua² , Chunyan Wang¹ , Qingtian Guan³ , Jing Wu¹ , Bing Liu² , Lei Song²

¹Department of General Practice, The First Hospital of Jilin University, ²Department of Respiratory Medicine, Center for Pathogen Biology and Infectious Diseases, The First Hospital of Jilin University, ³Bioinformatics Laboratory, The First Hospital of Jilin University

Summary

To facilitate the rapid and precise detection of Acinetobacter baumannii, we present a protocol that employs Recombinase Polymerase Amplification (RPA) in conjunction with LbaCas12a endonuclease for identifying A. baumannii infections.

Abstract

Acinetobacter baumannii, a gram-negative bacterium, is notorious for causing severe infections with high mortality rates. Rapid and accurate detection of A. baumannii is crucial for prompt treatment, effective infection control, and curbing antibiotic resistance. However, there is no suitable method for rapid and easy on-site detection of A. baumannii. The DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR) system offers a rapid, precise, and sensitive approach to A. baumannii detection by integrating the target-specific recognition capabilities of Cas12a with the isothermal amplification efficiency of Recombinase Polymerase Amplification (RPA). This protocol details the detection of A. baumannii using RPA combined with LbaCas12a endonuclease. The following steps are described in this article: extraction of DNA, selection of a specific DNA sequence, design of primer and CRISPR RNA (crRNA), construction of positive recombinant plasmid, setup of Cas12a-RPA assay, optimization of the RPA amplification system, visualization of the RPA-CRISPR/Cas12a assay using a fluorescence detection tool such as a real-time PCR instrument, and evaluation of sensitivity and specificity evaluation.

Introduction

In clinical microbiology, detecting Acinetobacter baumannii infections presents a significant challenge. This gram-negative bacterium can cause infections with severe clinical symptoms, even with high mortality rates, particularly among immunocompromised patients¹. Traditional culture-based detection methods for pathogen infections are time-consuming and may lack sensitivity, which may delay antibiotic treatment and compromise patient outcomes. Rapid and accurate identification of A. baumannii is crucial for effective treatment and outbreak control. Molecular techniques employing nucleic acid amplification have been explored t....