Rapid Bacterial Identification and Antimicrobial Susceptibility Testing Directly from Urine Samples via an Asymmetric Polymerase Chain Reaction-Cas12a Platform

作者：Laibao Zheng, Xinxi Zhou, Yan Zhang, Weiwei Wang, Chenlu Chen, Xuefeng Lin, Yan Zheng, Yongliang Lou · 发表于：Analytical Chemistry · 年份：2025 · DOI：10.1021/acs.analchem.5c04410 · 被引用次数：4 · 研究领域：Bacterial Identification and Susceptibility Testing、CRISPR and Genetic Engineering、Biosensors and Analytical Detection

Antimicrobial resistance poses a critical global health challenge, largely due to the prolonged turnaround times of conventional pathogen identification (ID) and antimicrobial susceptibility testing (AST). Here, we present a clinically validated diagnostic platform integrating asymmetric polymerase chain reaction (aPCR) with CRISPR/Cas12a for direct bacterial ID and phenotypic AST from urine samples. Unlike traditional multiplex PCR requiring complex primer sets, our platform employs a singleplex aPCR targeting the V3–V4 region of 16S rDNA to generate single-stranded and double-stranded DNA. This design enables protospacer adjacent motif-free activation of Cas12a when required via the ssDNA fraction generated by aPCR, facilitating species-level multiplex detection of six common uropathogens at 10 3 CFU/mL via programmable CRISPR/Cas12a crRNAs. Phenotypic AST is accomplished within 60 min by quantifying nucleic acid changes following antibiotic exposure, allowing accurate discrimination between susceptible and resistant strains. When validated with 86 clinical urine samples, the aPCR–Cas12a platform achieved complete concordance with culture-based identification among the 45 samples carrying target pathogens and demonstrated high accuracy for AST, confirming its reliability for direct pathogen detection and susceptibility assessment from urine. The complete workflow requires only 5.5 h, significantly reducing the diagnostic time compared to standard methods (>48 h). This rapid...