A one-pot, one-step CRISPR-AapCas12b-based platform for sensitive and specific detection of Klebsiella pneumoniae
Klebsiella pneumoniae (K. pneumoniae) was a prevalent nosocomial pathogen and a major cause of neonatal sepsis, highlighting the critical need for simple, rapid, sensitive and reliable diagnosis for K. pneumoniae infection. While CRISPR-based diagnostics provide a transformative technology to nuclei...
Saved in:
| Main Authors: | , , , , , , , , , , , , |
|---|---|
| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2025-06-01
|
| Series: | Sensing and Bio-Sensing Research |
| Subjects: | |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2214180425000637 |
| Tags: |
Add Tag
No Tags, Be the first to tag this record!
|
| _version_ | 1849335670436265984 |
|---|---|
| author | Juan Zhou Zirong Wu Lei Yu Xiaolan Huang Fei Xiao Yu Zhang Linglong Wan Jin Fu Nan Jia Min Chen Chunrong Sun Zhaomin Feng Yi Wang |
| author_facet | Juan Zhou Zirong Wu Lei Yu Xiaolan Huang Fei Xiao Yu Zhang Linglong Wan Jin Fu Nan Jia Min Chen Chunrong Sun Zhaomin Feng Yi Wang |
| author_sort | Juan Zhou |
| collection | DOAJ |
| description | Klebsiella pneumoniae (K. pneumoniae) was a prevalent nosocomial pathogen and a major cause of neonatal sepsis, highlighting the critical need for simple, rapid, sensitive and reliable diagnosis for K. pneumoniae infection. While CRISPR-based diagnostics provide a transformative technology to nucleic acid detection, current CRISPR-based methods often rely on PAM-dependent sequences, involve multiple steps, and are hampered by cumbersome manual operation, sequence limitations and the risk of aerosol contamination. To address these challenges, we developed a novel one-pot, one-step, PAM-independent CRISPR-Cas12b-based detection platform for rapid, sensitive and specific detection of K. pneumoniae, termed CRISPR-KP. This assay integrates multiple cross displacement amplification (MCDA) with CRISPR-Cas12b system in a single reaction mixture, employing MCDA for target nucleic acid amplification and the trans-cleavage activity of CRISPR-Cas12b system for amplification signal decoding. This one-pot assay achieves a remarkable limit of detection 10 fg (∼1.6 copies) of K. pneumoniae DNA within a 40-min isothermal reaction at 59 °C, eliminating the need for complex multi-step procedures. CRISPR-KP demonstrated comparable diagnostic efficiency to the clinical standard method in bronchoalveolar lavage fluid (BALF) samples, with reduced turnaround time and minimal manual operation, highlighting the excellent reliability and accuracy of our assay. Taken together, the CRISPR-KP assay offers a promising diagnostic tool for K. pneumoniae detection in centralized laboratories, point-of-care settings, and primary healthcare institutions. |
| format | Article |
| id | doaj-art-52c424e75bf64989abec7646f4027427 |
| institution | Kabale University |
| issn | 2214-1804 |
| language | English |
| publishDate | 2025-06-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Sensing and Bio-Sensing Research |
| spelling | doaj-art-52c424e75bf64989abec7646f40274272025-08-20T03:45:11ZengElsevierSensing and Bio-Sensing Research2214-18042025-06-014810079710.1016/j.sbsr.2025.100797A one-pot, one-step CRISPR-AapCas12b-based platform for sensitive and specific detection of Klebsiella pneumoniaeJuan Zhou0Zirong Wu1Lei Yu2Xiaolan Huang3Fei Xiao4Yu Zhang5Linglong Wan6Jin Fu7Nan Jia8Min Chen9Chunrong Sun10Zhaomin Feng11Yi Wang12Experimental Research Center, Capital Center for Children's Health, Capital Medical University, Capital Institute of Pediatrics, Beijing 100020, PR ChinaDepartment of Clinical Laboratory, Xiangyang No. 1 People's Hospital, Hubei University of Medicine, Xiangyang, PR ChinaDepartment of Infection Management, Capital Center for Children's Health, Capital Medical University, Beijing 100020, PR ChinaExperimental Research Center, Capital Center for Children's Health, Capital Medical University, Capital Institute of Pediatrics, Beijing 100020, PR ChinaExperimental Research Center, Capital Center for Children's Health, Capital Medical University, Capital Institute of Pediatrics, Beijing 100020, PR China; Molecular Diagnostic Center, Capital Center for Children's Health, Capital Medical University, Beijing 100020, PR ChinaExperimental Research Center, Capital Center for Children's Health, Capital Medical University, Capital Institute of Pediatrics, Beijing 100020, PR ChinaExperimental Research Center, Capital Center for Children's Health, Capital Medical University, Capital Institute of Pediatrics, Beijing 100020, PR ChinaExperimental Research Center, Capital Center for Children's Health, Capital Medical University, Capital Institute of Pediatrics, Beijing 100020, PR ChinaExperimental Research Center, Capital Center for Children's Health, Capital Medical University, Capital Institute of Pediatrics, Beijing 100020, PR ChinaExperimental Research Center, Capital Center for Children's Health, Capital Medical University, Capital Institute of Pediatrics, Beijing 100020, PR ChinaExperimental Research Center, Capital Center for Children's Health, Capital Medical University, Capital Institute of Pediatrics, Beijing 100020, PR ChinaBeijing Key Laboratory of Surveillance, Early Warning and Pathogen Research on Emerging Infectious Diseases, Beijing Center for Disease Prevention and Control, Beijing 100013, China; Beijing Research Center for Respiratory Infectious Diseases, Beijing 100013, PR China; Correspondence to: Z. Feng, Beijing Center for Disease Prevention and Control, Beijing 100013, PR China.Experimental Research Center, Capital Center for Children's Health, Capital Medical University, Capital Institute of Pediatrics, Beijing 100020, PR China; Molecular Diagnostic Center, Capital Center for Children's Health, Capital Medical University, Beijing 100020, PR China; Correspondence to: Y. Wang, Experimental Research Center, Capital Center for Children's Health, Capital Medical University, Capital Institute of Pediatrics, Beijing 100020, PR China.Klebsiella pneumoniae (K. pneumoniae) was a prevalent nosocomial pathogen and a major cause of neonatal sepsis, highlighting the critical need for simple, rapid, sensitive and reliable diagnosis for K. pneumoniae infection. While CRISPR-based diagnostics provide a transformative technology to nucleic acid detection, current CRISPR-based methods often rely on PAM-dependent sequences, involve multiple steps, and are hampered by cumbersome manual operation, sequence limitations and the risk of aerosol contamination. To address these challenges, we developed a novel one-pot, one-step, PAM-independent CRISPR-Cas12b-based detection platform for rapid, sensitive and specific detection of K. pneumoniae, termed CRISPR-KP. This assay integrates multiple cross displacement amplification (MCDA) with CRISPR-Cas12b system in a single reaction mixture, employing MCDA for target nucleic acid amplification and the trans-cleavage activity of CRISPR-Cas12b system for amplification signal decoding. This one-pot assay achieves a remarkable limit of detection 10 fg (∼1.6 copies) of K. pneumoniae DNA within a 40-min isothermal reaction at 59 °C, eliminating the need for complex multi-step procedures. CRISPR-KP demonstrated comparable diagnostic efficiency to the clinical standard method in bronchoalveolar lavage fluid (BALF) samples, with reduced turnaround time and minimal manual operation, highlighting the excellent reliability and accuracy of our assay. Taken together, the CRISPR-KP assay offers a promising diagnostic tool for K. pneumoniae detection in centralized laboratories, point-of-care settings, and primary healthcare institutions.http://www.sciencedirect.com/science/article/pii/S2214180425000637K. pneumoniaeMultiple cross displacement amplificationCRISPR-KPAapCas12bOne-step |
| spellingShingle | Juan Zhou Zirong Wu Lei Yu Xiaolan Huang Fei Xiao Yu Zhang Linglong Wan Jin Fu Nan Jia Min Chen Chunrong Sun Zhaomin Feng Yi Wang A one-pot, one-step CRISPR-AapCas12b-based platform for sensitive and specific detection of Klebsiella pneumoniae Sensing and Bio-Sensing Research K. pneumoniae Multiple cross displacement amplification CRISPR-KP AapCas12b One-step |
| title | A one-pot, one-step CRISPR-AapCas12b-based platform for sensitive and specific detection of Klebsiella pneumoniae |
| title_full | A one-pot, one-step CRISPR-AapCas12b-based platform for sensitive and specific detection of Klebsiella pneumoniae |
| title_fullStr | A one-pot, one-step CRISPR-AapCas12b-based platform for sensitive and specific detection of Klebsiella pneumoniae |
| title_full_unstemmed | A one-pot, one-step CRISPR-AapCas12b-based platform for sensitive and specific detection of Klebsiella pneumoniae |
| title_short | A one-pot, one-step CRISPR-AapCas12b-based platform for sensitive and specific detection of Klebsiella pneumoniae |
| title_sort | one pot one step crispr aapcas12b based platform for sensitive and specific detection of klebsiella pneumoniae |
| topic | K. pneumoniae Multiple cross displacement amplification CRISPR-KP AapCas12b One-step |
| url | http://www.sciencedirect.com/science/article/pii/S2214180425000637 |
| work_keys_str_mv | AT juanzhou aonepotonestepcrispraapcas12bbasedplatformforsensitiveandspecificdetectionofklebsiellapneumoniae AT zirongwu aonepotonestepcrispraapcas12bbasedplatformforsensitiveandspecificdetectionofklebsiellapneumoniae AT leiyu aonepotonestepcrispraapcas12bbasedplatformforsensitiveandspecificdetectionofklebsiellapneumoniae AT xiaolanhuang aonepotonestepcrispraapcas12bbasedplatformforsensitiveandspecificdetectionofklebsiellapneumoniae AT feixiao aonepotonestepcrispraapcas12bbasedplatformforsensitiveandspecificdetectionofklebsiellapneumoniae AT yuzhang aonepotonestepcrispraapcas12bbasedplatformforsensitiveandspecificdetectionofklebsiellapneumoniae AT linglongwan aonepotonestepcrispraapcas12bbasedplatformforsensitiveandspecificdetectionofklebsiellapneumoniae AT jinfu aonepotonestepcrispraapcas12bbasedplatformforsensitiveandspecificdetectionofklebsiellapneumoniae AT nanjia aonepotonestepcrispraapcas12bbasedplatformforsensitiveandspecificdetectionofklebsiellapneumoniae AT minchen aonepotonestepcrispraapcas12bbasedplatformforsensitiveandspecificdetectionofklebsiellapneumoniae AT chunrongsun aonepotonestepcrispraapcas12bbasedplatformforsensitiveandspecificdetectionofklebsiellapneumoniae AT zhaominfeng aonepotonestepcrispraapcas12bbasedplatformforsensitiveandspecificdetectionofklebsiellapneumoniae AT yiwang aonepotonestepcrispraapcas12bbasedplatformforsensitiveandspecificdetectionofklebsiellapneumoniae AT juanzhou onepotonestepcrispraapcas12bbasedplatformforsensitiveandspecificdetectionofklebsiellapneumoniae AT zirongwu onepotonestepcrispraapcas12bbasedplatformforsensitiveandspecificdetectionofklebsiellapneumoniae AT leiyu onepotonestepcrispraapcas12bbasedplatformforsensitiveandspecificdetectionofklebsiellapneumoniae AT xiaolanhuang onepotonestepcrispraapcas12bbasedplatformforsensitiveandspecificdetectionofklebsiellapneumoniae AT feixiao onepotonestepcrispraapcas12bbasedplatformforsensitiveandspecificdetectionofklebsiellapneumoniae AT yuzhang onepotonestepcrispraapcas12bbasedplatformforsensitiveandspecificdetectionofklebsiellapneumoniae AT linglongwan onepotonestepcrispraapcas12bbasedplatformforsensitiveandspecificdetectionofklebsiellapneumoniae AT jinfu onepotonestepcrispraapcas12bbasedplatformforsensitiveandspecificdetectionofklebsiellapneumoniae AT nanjia onepotonestepcrispraapcas12bbasedplatformforsensitiveandspecificdetectionofklebsiellapneumoniae AT minchen onepotonestepcrispraapcas12bbasedplatformforsensitiveandspecificdetectionofklebsiellapneumoniae AT chunrongsun onepotonestepcrispraapcas12bbasedplatformforsensitiveandspecificdetectionofklebsiellapneumoniae AT zhaominfeng onepotonestepcrispraapcas12bbasedplatformforsensitiveandspecificdetectionofklebsiellapneumoniae AT yiwang onepotonestepcrispraapcas12bbasedplatformforsensitiveandspecificdetectionofklebsiellapneumoniae |