Rapid, highly specific and sensitive detection of human adenovirus using multiple cross displacement amplification coupled with CRISPR-Cas12a-based detection
Human adenoviruses (HAdV), particularly serotypes 3 and 7, are significant pathogens that cause respiratory tract infection. Here, we developed a novel HAdV diagnostic assay, termed HAdV-MCDA-CRISPR, by combining multiple cross displacement amplification (MCDA) with CRISPR/Cas12a-based detection. HA...
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Elsevier
2025-08-01
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S2666831925000645 |
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| author | Linglong Wan Juan Zhou Lei Yu Xiaolan Huang Jin Fu Fei Xiao Nan Jia Yu Zhang Min Chen Xu Chen Zhaomin Feng Yi Wang Lihui Meng |
| author_facet | Linglong Wan Juan Zhou Lei Yu Xiaolan Huang Jin Fu Fei Xiao Nan Jia Yu Zhang Min Chen Xu Chen Zhaomin Feng Yi Wang Lihui Meng |
| author_sort | Linglong Wan |
| collection | DOAJ |
| description | Human adenoviruses (HAdV), particularly serotypes 3 and 7, are significant pathogens that cause respiratory tract infection. Here, we developed a novel HAdV diagnostic assay, termed HAdV-MCDA-CRISPR, by combining multiple cross displacement amplification (MCDA) with CRISPR/Cas12a-based detection. HAdV-MCDA-CRISPR assay uses MCDA to amplify target DNA, followed by detection of predefined sequences using a CRISPR/Cas12a-crRNA complex. Resulting fluorescence from probe cleavage indicates a positive result. A set of 10 MCDA primers and a specific crRNA were designed to ensure high specificity of HAdV-MCDA-CRISPR assay. The entire procedure, including rapid template preparation (∼15 min), MCDA reaction (40 min), and result interpretation (5 min), takes approximately one hour. Targeting the conserved hexon gene, HAdV-MCDA-CRISPR achieves high sensitivity under optimized MCDA reaction conditions (40 min at 66 °C), detecting as little as 1.92 copies/μL of hexon-containing plasmid DNA without cross-reactivity. Validation using 128 clinical samples demonstrated 78/80 positive results for HAdV-positive patients and 48/48 negative results for HAdV-negative patients. Thus, HAdV-MCDA-CRISPR assay offers a promising alternative for rapid and accurate clinical diagnosis of HAdV-3 and HAdV-7 infections. |
| format | Article |
| id | doaj-art-a2ae8b87928449daa60fdea6bcc6089f |
| institution | DOAJ |
| issn | 2666-8319 |
| language | English |
| publishDate | 2025-08-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Talanta Open |
| spelling | doaj-art-a2ae8b87928449daa60fdea6bcc6089f2025-08-20T02:55:20ZengElsevierTalanta Open2666-83192025-08-011110046210.1016/j.talo.2025.100462Rapid, highly specific and sensitive detection of human adenovirus using multiple cross displacement amplification coupled with CRISPR-Cas12a-based detectionLinglong Wan0Juan Zhou1Lei Yu2Xiaolan Huang3Jin Fu4Fei Xiao5Nan Jia6Yu Zhang7Min Chen8Xu Chen9Zhaomin Feng10Yi Wang11Lihui Meng12Experimental 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 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 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 ChinaThe Second Clinical Medical College, Guizhou University of Traditional Chinese Medicine, Guiyang, Guizhou 550003, PR China; Department of Scientific Research, The Second Affiliated Hospital, Guizhou University of Traditional Chinese Medicine, Guiyang, Guizhou 550003, PR China; Corresponding authors.Beijing 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, China; Corresponding authors.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; Corresponding authors.Department of Infectious Diseases, Capital Center for Children's Health, Capital Medical University, Beijing 100020, PR China; Corresponding authors.Human adenoviruses (HAdV), particularly serotypes 3 and 7, are significant pathogens that cause respiratory tract infection. Here, we developed a novel HAdV diagnostic assay, termed HAdV-MCDA-CRISPR, by combining multiple cross displacement amplification (MCDA) with CRISPR/Cas12a-based detection. HAdV-MCDA-CRISPR assay uses MCDA to amplify target DNA, followed by detection of predefined sequences using a CRISPR/Cas12a-crRNA complex. Resulting fluorescence from probe cleavage indicates a positive result. A set of 10 MCDA primers and a specific crRNA were designed to ensure high specificity of HAdV-MCDA-CRISPR assay. The entire procedure, including rapid template preparation (∼15 min), MCDA reaction (40 min), and result interpretation (5 min), takes approximately one hour. Targeting the conserved hexon gene, HAdV-MCDA-CRISPR achieves high sensitivity under optimized MCDA reaction conditions (40 min at 66 °C), detecting as little as 1.92 copies/μL of hexon-containing plasmid DNA without cross-reactivity. Validation using 128 clinical samples demonstrated 78/80 positive results for HAdV-positive patients and 48/48 negative results for HAdV-negative patients. Thus, HAdV-MCDA-CRISPR assay offers a promising alternative for rapid and accurate clinical diagnosis of HAdV-3 and HAdV-7 infections.http://www.sciencedirect.com/science/article/pii/S2666831925000645Human adenovirusesMultiple cross displacement amplificationCRISPR, Cas12a, crRNA |
| spellingShingle | Linglong Wan Juan Zhou Lei Yu Xiaolan Huang Jin Fu Fei Xiao Nan Jia Yu Zhang Min Chen Xu Chen Zhaomin Feng Yi Wang Lihui Meng Rapid, highly specific and sensitive detection of human adenovirus using multiple cross displacement amplification coupled with CRISPR-Cas12a-based detection Talanta Open Human adenoviruses Multiple cross displacement amplification CRISPR, Cas12a, crRNA |
| title | Rapid, highly specific and sensitive detection of human adenovirus using multiple cross displacement amplification coupled with CRISPR-Cas12a-based detection |
| title_full | Rapid, highly specific and sensitive detection of human adenovirus using multiple cross displacement amplification coupled with CRISPR-Cas12a-based detection |
| title_fullStr | Rapid, highly specific and sensitive detection of human adenovirus using multiple cross displacement amplification coupled with CRISPR-Cas12a-based detection |
| title_full_unstemmed | Rapid, highly specific and sensitive detection of human adenovirus using multiple cross displacement amplification coupled with CRISPR-Cas12a-based detection |
| title_short | Rapid, highly specific and sensitive detection of human adenovirus using multiple cross displacement amplification coupled with CRISPR-Cas12a-based detection |
| title_sort | rapid highly specific and sensitive detection of human adenovirus using multiple cross displacement amplification coupled with crispr cas12a based detection |
| topic | Human adenoviruses Multiple cross displacement amplification CRISPR, Cas12a, crRNA |
| url | http://www.sciencedirect.com/science/article/pii/S2666831925000645 |
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