Establishment and optimization of a system for the detection of Candida albicans based on enzymatic recombinase amplification and CRISPR/Cas12a system
ABSTRACT Invasive candidiasis is a fungal infection caused by various pathogenic yeasts, with Candida albicans as the predominant pathogen. Traditional culturing and identification methods for C. albicans are slow, requiring several days to weeks to produce results, which hampers rapid diagnosis. In...
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| Language: | English |
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American Society for Microbiology
2025-05-01
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| Series: | Microbiology Spectrum |
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| Online Access: | https://journals.asm.org/doi/10.1128/spectrum.00268-25 |
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| author | Xiaotong Zeng Qiuyang Jiang Fo Yang Qianlin Wu Tingyao lyu Qi Zhang Jin Wang Feng Li Dayong Xu |
| author_facet | Xiaotong Zeng Qiuyang Jiang Fo Yang Qianlin Wu Tingyao lyu Qi Zhang Jin Wang Feng Li Dayong Xu |
| author_sort | Xiaotong Zeng |
| collection | DOAJ |
| description | ABSTRACT Invasive candidiasis is a fungal infection caused by various pathogenic yeasts, with Candida albicans as the predominant pathogen. Traditional culturing and identification methods for C. albicans are slow, requiring several days to weeks to produce results, which hampers rapid diagnosis. In this study, we proposed three amplification methods to combine with CRISPR/Cas12a and selected the enzymatic recombinase amplification (ERA) and CRISPR/Cas12a two-step method for the detection of C. albicans in terms of sensitivity, and then the two-step method was optimized to a temperature-controlled one-step method for the detection of C. albicans by enzymatic recombinase amplification (ERA)-CRISPR/Cas12a. The temperature-controlled system employs a combination of liquid and solid paraffin wax to maintain the desired melting point, thus facilitating spatial separation of the ERA amplification system from the CRISPR/Cas12a detection system within a single tube. After a reaction at 37°C, the temperature is raised to 45°C, melting the wax and allowing the amplification system to merge with the detection system, initiating the reaction. This one-step detection platform simplifies and expedites the procedure, achieving a sensitivity level on par with that of two-step methods. The reaction completes in about 30 minutes, detecting as little as 100 ag/µL of genomic DNA from C. albicans pure cultures. It shows high specificity and resistance to clinical nucleic acid interference, without cross-reactivity. Additionally, the method eliminates the need to open the reaction tube, effectively preventing aerosol contamination and providing a stable, thus offering a new tool for the rapid clinical diagnosis of C. albicans.IMPORTANCEThis study established a two-step method through optimization, compared its sensitivity, and then combined the specific detection capabilities of ERA and CRISPR/Cas12a. Furthermore, a one-step method was developed based on the two-step method, creating a one-step system for the detection of Candida albicans. This system does not require the lid to be opened during the reaction process, reducing aerosol contamination and minimizing the risk of false positives. This method does not require advanced instruments or equipment and shows strong specificity without being affected by other pathogens. It can serve as a new method for the detection of Candida albicans and has significant practical application prospects. |
| format | Article |
| id | doaj-art-774d71c04b624cd2a3adac6b6060c393 |
| institution | OA Journals |
| issn | 2165-0497 |
| language | English |
| publishDate | 2025-05-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | Microbiology Spectrum |
| spelling | doaj-art-774d71c04b624cd2a3adac6b6060c3932025-08-20T02:14:44ZengAmerican Society for MicrobiologyMicrobiology Spectrum2165-04972025-05-0113510.1128/spectrum.00268-25Establishment and optimization of a system for the detection of Candida albicans based on enzymatic recombinase amplification and CRISPR/Cas12a systemXiaotong Zeng0Qiuyang Jiang1Fo Yang2Qianlin Wu3Tingyao lyu4Qi Zhang5Jin Wang6Feng Li7Dayong Xu8Anhui Province Key Laboratory of Pollutant Sensitive Materials and Environmental Remediation, Huaibei Normal University, Huaibei, Anhui, ChinaAnhui Province Key Laboratory of Pollutant Sensitive Materials and Environmental Remediation, Huaibei Normal University, Huaibei, Anhui, ChinaAnhui Province Key Laboratory of Pollutant Sensitive Materials and Environmental Remediation, Huaibei Normal University, Huaibei, Anhui, ChinaAnhui Province Key Laboratory of Pollutant Sensitive Materials and Environmental Remediation, Huaibei Normal University, Huaibei, Anhui, ChinaAnhui Province Key Laboratory of Pollutant Sensitive Materials and Environmental Remediation, Huaibei Normal University, Huaibei, Anhui, ChinaHuaibei People’s Hospital, Huaibei, Anhui, ChinaAnhui Province Key Laboratory of Pollutant Sensitive Materials and Environmental Remediation, Huaibei Normal University, Huaibei, Anhui, ChinaAnhui Province Key Laboratory of Pollutant Sensitive Materials and Environmental Remediation, Huaibei Normal University, Huaibei, Anhui, ChinaAnhui Province Key Laboratory of Pollutant Sensitive Materials and Environmental Remediation, Huaibei Normal University, Huaibei, Anhui, ChinaABSTRACT Invasive candidiasis is a fungal infection caused by various pathogenic yeasts, with Candida albicans as the predominant pathogen. Traditional culturing and identification methods for C. albicans are slow, requiring several days to weeks to produce results, which hampers rapid diagnosis. In this study, we proposed three amplification methods to combine with CRISPR/Cas12a and selected the enzymatic recombinase amplification (ERA) and CRISPR/Cas12a two-step method for the detection of C. albicans in terms of sensitivity, and then the two-step method was optimized to a temperature-controlled one-step method for the detection of C. albicans by enzymatic recombinase amplification (ERA)-CRISPR/Cas12a. The temperature-controlled system employs a combination of liquid and solid paraffin wax to maintain the desired melting point, thus facilitating spatial separation of the ERA amplification system from the CRISPR/Cas12a detection system within a single tube. After a reaction at 37°C, the temperature is raised to 45°C, melting the wax and allowing the amplification system to merge with the detection system, initiating the reaction. This one-step detection platform simplifies and expedites the procedure, achieving a sensitivity level on par with that of two-step methods. The reaction completes in about 30 minutes, detecting as little as 100 ag/µL of genomic DNA from C. albicans pure cultures. It shows high specificity and resistance to clinical nucleic acid interference, without cross-reactivity. Additionally, the method eliminates the need to open the reaction tube, effectively preventing aerosol contamination and providing a stable, thus offering a new tool for the rapid clinical diagnosis of C. albicans.IMPORTANCEThis study established a two-step method through optimization, compared its sensitivity, and then combined the specific detection capabilities of ERA and CRISPR/Cas12a. Furthermore, a one-step method was developed based on the two-step method, creating a one-step system for the detection of Candida albicans. This system does not require the lid to be opened during the reaction process, reducing aerosol contamination and minimizing the risk of false positives. This method does not require advanced instruments or equipment and shows strong specificity without being affected by other pathogens. It can serve as a new method for the detection of Candida albicans and has significant practical application prospects.https://journals.asm.org/doi/10.1128/spectrum.00268-25Candida albicansERACRISPR/Cas12a |
| spellingShingle | Xiaotong Zeng Qiuyang Jiang Fo Yang Qianlin Wu Tingyao lyu Qi Zhang Jin Wang Feng Li Dayong Xu Establishment and optimization of a system for the detection of Candida albicans based on enzymatic recombinase amplification and CRISPR/Cas12a system Microbiology Spectrum Candida albicans ERA CRISPR/Cas12a |
| title | Establishment and optimization of a system for the detection of Candida albicans based on enzymatic recombinase amplification and CRISPR/Cas12a system |
| title_full | Establishment and optimization of a system for the detection of Candida albicans based on enzymatic recombinase amplification and CRISPR/Cas12a system |
| title_fullStr | Establishment and optimization of a system for the detection of Candida albicans based on enzymatic recombinase amplification and CRISPR/Cas12a system |
| title_full_unstemmed | Establishment and optimization of a system for the detection of Candida albicans based on enzymatic recombinase amplification and CRISPR/Cas12a system |
| title_short | Establishment and optimization of a system for the detection of Candida albicans based on enzymatic recombinase amplification and CRISPR/Cas12a system |
| title_sort | establishment and optimization of a system for the detection of candida albicans based on enzymatic recombinase amplification and crispr cas12a system |
| topic | Candida albicans ERA CRISPR/Cas12a |
| url | https://journals.asm.org/doi/10.1128/spectrum.00268-25 |
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