Development of a Recombinase Polymerase Amplification-Coupled CRISPR/Cas12a Platform for Rapid Detection of Antimicrobial-Resistant Genes in Carbapenem-Resistant <i>Enterobacterales</i>
The worldwide spread of carbapenemase-producing <i>Enterobacterales</i> (CPE) represents a significant threat owing to the high mortality and morbidity rates. Traditional diagnostic methods are often too slow and complex for rapid point-of-care testing. Therefore, we developed a recombin...
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| Main Authors: | , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
MDPI AG
2024-11-01
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| Series: | Biosensors |
| Subjects: | |
| Online Access: | https://www.mdpi.com/2079-6374/14/11/536 |
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| Summary: | The worldwide spread of carbapenemase-producing <i>Enterobacterales</i> (CPE) represents a significant threat owing to the high mortality and morbidity rates. Traditional diagnostic methods are often too slow and complex for rapid point-of-care testing. Therefore, we developed a recombinase polymerase amplification (RPA)-coupled CRISPR/Cas12a system (RCCS), a rapid, accurate, and simple diagnostic platform for detecting antimicrobial-resistant genes. The RCCS detected carbapenemase genes (<i>bla</i><sub>KPC</sub> and <i>bla</i><sub>NDM</sub>) within 50 min, including 10 min for DNA extraction and 30–40 min for RCCS reaction (a 20 min RPA reaction with a 10–20-min CRISPR/Cas12a assay). Fluorescence signals obtained from the RCCS platform were visualized using lateral-flow test strips (LFSs) and real-time and endpoint fluorescence. The LFS clearly displayed test lines while detecting carbapenemase genes. Furthermore, the RCCS platform demonstrated high sensitivity by successfully detecting <i>bla</i><sub>KPC</sub> and <i>bla</i><sub>NDM</sub> at the attomolar and picomolar levels, respectively. The accuracy of the RCCS platform was validated with clinical isolates of <i>Klebsiella pneumoniae</i> and <i>Escherichia coli</i>; a 100% detection accuracy was achieved, which has not been reported when using conventional PCR. Overall, these findings indicate that the RCCS platform is a powerful tool for rapid and reliable detection of carbapenemase-encoding genes, with significant potential for implementation in point-of-care settings and resource-limited environments. |
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| ISSN: | 2079-6374 |