Innovative nucleic acid detection of Clostridioides difficile utilizing the PAM-unconventional, one-step LAMP/CRISPR-Cas12b detection platforms
IntroductionClostridioides difficile (C. difficile), a human pathogen that causes diarrhea and colon lesions, has garnered widespread attention. Rapid and accurate detection of bacterial virulence factors is essential for the diagnosis of C. difficile infection (CDI). To date, numerous laboratory te...
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Frontiers Media S.A.
2025-05-01
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| Series: | Frontiers in Cellular and Infection Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fcimb.2025.1594271/full |
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| author | Yizhuo Zhang Luqin Lv Su Xu Yijian Chen |
| author_facet | Yizhuo Zhang Luqin Lv Su Xu Yijian Chen |
| author_sort | Yizhuo Zhang |
| collection | DOAJ |
| description | IntroductionClostridioides difficile (C. difficile), a human pathogen that causes diarrhea and colon lesions, has garnered widespread attention. Rapid and accurate detection of bacterial virulence factors is essential for the diagnosis of C. difficile infection (CDI). To date, numerous laboratory tests have been developed; however, none fully meet the combined requirements of speed, cost-effectiveness, portability, sensitivity, and specificity. Molecular diagnostic technologies based on CRISPR-Cas systems have provided a promising solution to this challenge. Nonetheless, the limited compatibility between pre-amplification and CRISPR cleavage, coupled with the inherent selectivity of CRISPR systems for protospacer adjacent motif (PAM) sequences near the target site, poses additional constraints on the broader adoption of this approach.MethodsHere, we developed PAM-unconventional, One-step LAMP/CRISPR-Cas12b (POLC) detection platforms for the toxin-encoding genes tcdA and tcdB of C. difficile.ResultsThe POLC platforms operated at 60 °C, enabling result interpretation either through fluorescence intensity measurements or direct visualization under UV light. The limits of detection (LoDs) ranged from 3 to 14 copies/μL using a fluorescence reader and from 6 to 18 copies/μL via direct observation. Compared to qPCR, which typically requires over an hour, the POLC platforms reduced the detection time to approximately 40 minutes. Each reaction cost approximately USD 6.5, offering a substantial cost saving compared to qPCR-based commercial kits (over USD 10 per test). In clinical validation with 55 fecal samples, the tcdA POLC assay achieved 86.4% sensitivity and 84.8% specificity, while the tcdB POLC assay demonstrated 96.6% sensitivity and 100% specificity, using qPCR as the reference standard.DiscussionOur research presents innovative CRISPR-based one-step nucleic acid detection platforms that eliminate canonical PAM sequence requirements. These platforms exhibit high sensitivity and specificity while achieving rapid detection under simple conditions, making them promising candidates for clinical diagnostics and point-of-care testing (POCT). |
| format | Article |
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| language | English |
| publishDate | 2025-05-01 |
| publisher | Frontiers Media S.A. |
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| series | Frontiers in Cellular and Infection Microbiology |
| spelling | doaj-art-773c8a12527f4e5595308b4787ccc28b2025-08-20T02:01:20ZengFrontiers Media S.A.Frontiers in Cellular and Infection Microbiology2235-29882025-05-011510.3389/fcimb.2025.15942711594271Innovative nucleic acid detection of Clostridioides difficile utilizing the PAM-unconventional, one-step LAMP/CRISPR-Cas12b detection platformsYizhuo ZhangLuqin LvSu XuYijian ChenIntroductionClostridioides difficile (C. difficile), a human pathogen that causes diarrhea and colon lesions, has garnered widespread attention. Rapid and accurate detection of bacterial virulence factors is essential for the diagnosis of C. difficile infection (CDI). To date, numerous laboratory tests have been developed; however, none fully meet the combined requirements of speed, cost-effectiveness, portability, sensitivity, and specificity. Molecular diagnostic technologies based on CRISPR-Cas systems have provided a promising solution to this challenge. Nonetheless, the limited compatibility between pre-amplification and CRISPR cleavage, coupled with the inherent selectivity of CRISPR systems for protospacer adjacent motif (PAM) sequences near the target site, poses additional constraints on the broader adoption of this approach.MethodsHere, we developed PAM-unconventional, One-step LAMP/CRISPR-Cas12b (POLC) detection platforms for the toxin-encoding genes tcdA and tcdB of C. difficile.ResultsThe POLC platforms operated at 60 °C, enabling result interpretation either through fluorescence intensity measurements or direct visualization under UV light. The limits of detection (LoDs) ranged from 3 to 14 copies/μL using a fluorescence reader and from 6 to 18 copies/μL via direct observation. Compared to qPCR, which typically requires over an hour, the POLC platforms reduced the detection time to approximately 40 minutes. Each reaction cost approximately USD 6.5, offering a substantial cost saving compared to qPCR-based commercial kits (over USD 10 per test). In clinical validation with 55 fecal samples, the tcdA POLC assay achieved 86.4% sensitivity and 84.8% specificity, while the tcdB POLC assay demonstrated 96.6% sensitivity and 100% specificity, using qPCR as the reference standard.DiscussionOur research presents innovative CRISPR-based one-step nucleic acid detection platforms that eliminate canonical PAM sequence requirements. These platforms exhibit high sensitivity and specificity while achieving rapid detection under simple conditions, making them promising candidates for clinical diagnostics and point-of-care testing (POCT).https://www.frontiersin.org/articles/10.3389/fcimb.2025.1594271/fullClostridioides difficileLAMPCRISPR-Cas12bnucleic acid detectionpoint-of-care testing |
| spellingShingle | Yizhuo Zhang Luqin Lv Su Xu Yijian Chen Innovative nucleic acid detection of Clostridioides difficile utilizing the PAM-unconventional, one-step LAMP/CRISPR-Cas12b detection platforms Frontiers in Cellular and Infection Microbiology Clostridioides difficile LAMP CRISPR-Cas12b nucleic acid detection point-of-care testing |
| title | Innovative nucleic acid detection of Clostridioides difficile utilizing the PAM-unconventional, one-step LAMP/CRISPR-Cas12b detection platforms |
| title_full | Innovative nucleic acid detection of Clostridioides difficile utilizing the PAM-unconventional, one-step LAMP/CRISPR-Cas12b detection platforms |
| title_fullStr | Innovative nucleic acid detection of Clostridioides difficile utilizing the PAM-unconventional, one-step LAMP/CRISPR-Cas12b detection platforms |
| title_full_unstemmed | Innovative nucleic acid detection of Clostridioides difficile utilizing the PAM-unconventional, one-step LAMP/CRISPR-Cas12b detection platforms |
| title_short | Innovative nucleic acid detection of Clostridioides difficile utilizing the PAM-unconventional, one-step LAMP/CRISPR-Cas12b detection platforms |
| title_sort | innovative nucleic acid detection of clostridioides difficile utilizing the pam unconventional one step lamp crispr cas12b detection platforms |
| topic | Clostridioides difficile LAMP CRISPR-Cas12b nucleic acid detection point-of-care testing |
| url | https://www.frontiersin.org/articles/10.3389/fcimb.2025.1594271/full |
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