Detection of Burkholderia pseudomallei with CRISPR-Cas12a based on specific sequence tags
Melioidosis is a bacterial infection caused by Burkholderia pseudomallei (B. pseudomallei), posing a significant threat to public health. Rapid and accurate detection of B. pseudomallei is crucial for preventing and controlling melioidosis. However, identifying B. pseudomallei is challenging due to...
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| Format: | Article |
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Frontiers Media S.A.
2023-04-01
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| Series: | Frontiers in Public Health |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fpubh.2023.1153352/full |
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| author | Jia-Xin Zhang Jian-Hao Xu Bing Yuan Xiao-Dong Wang Xu-hu Mao Jing-Lin Wang Xiang-Li-Lan Zhang Yuan Yuan |
| author_facet | Jia-Xin Zhang Jian-Hao Xu Bing Yuan Xiao-Dong Wang Xu-hu Mao Jing-Lin Wang Xiang-Li-Lan Zhang Yuan Yuan |
| author_sort | Jia-Xin Zhang |
| collection | DOAJ |
| description | Melioidosis is a bacterial infection caused by Burkholderia pseudomallei (B. pseudomallei), posing a significant threat to public health. Rapid and accurate detection of B. pseudomallei is crucial for preventing and controlling melioidosis. However, identifying B. pseudomallei is challenging due to its high similarity to other species in the same genus. To address this issue, this study proposed a dual-target method that can specifically identify B. pseudomallei in less than 40 min. We analyzed 1722 B. pseudomallei genomes to construct large-scale pan-genomes and selected specific sequence tags in their core genomes that effectively distinguish B. pseudomallei from its closely related species. Specifically, we selected two specific tags, LC1 and LC2, which we combined with the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated proteins (Cas12a) system and recombinase polymerase amplification (RPA) pre-amplification. Our analysis showed that the dual-target RPA-CRISPR/Cas12a assay has a sensitivity of approximately 0.2 copies/reaction and 10 fg genomic DNA for LC1, and 2 copies/reaction and 20 fg genomic DNA for LC2. Additionally, our method can accurately and rapidly detect B. pseudomallei in human blood and moist soil samples using the specific sequence tags mentioned above. In conclusion, the dual-target RPA-CRISPR/Cas12a method is a valuable tool for the rapid and accurate identification of B. pseudomallei in clinical and environmental samples, aiding in the prevention and control of melioidosis. |
| first_indexed | 2024-04-09T13:17:32Z |
| format | Article |
| id | doaj.art-22326693e6894948a83f84349797ebb1 |
| institution | Directory Open Access Journal |
| issn | 2296-2565 |
| language | English |
| last_indexed | 2024-04-09T13:17:32Z |
| publishDate | 2023-04-01 |
| publisher | Frontiers Media S.A. |
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| series | Frontiers in Public Health |
| spelling | doaj.art-22326693e6894948a83f84349797ebb12023-05-11T15:56:54ZengFrontiers Media S.A.Frontiers in Public Health2296-25652023-04-011110.3389/fpubh.2023.11533521153352Detection of Burkholderia pseudomallei with CRISPR-Cas12a based on specific sequence tagsJia-Xin Zhang0Jian-Hao Xu1Bing Yuan2Xiao-Dong Wang3Xu-hu Mao4Jing-Lin Wang5Xiang-Li-Lan Zhang6Yuan Yuan7State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, ChinaState Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, ChinaState Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, ChinaSchool of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, Fujian, ChinaDepartment of Clinical Microbiology and Immunology, The Third Military Medical University, Chongqing, ChinaState Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, ChinaState Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, ChinaState Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Beijing, ChinaMelioidosis is a bacterial infection caused by Burkholderia pseudomallei (B. pseudomallei), posing a significant threat to public health. Rapid and accurate detection of B. pseudomallei is crucial for preventing and controlling melioidosis. However, identifying B. pseudomallei is challenging due to its high similarity to other species in the same genus. To address this issue, this study proposed a dual-target method that can specifically identify B. pseudomallei in less than 40 min. We analyzed 1722 B. pseudomallei genomes to construct large-scale pan-genomes and selected specific sequence tags in their core genomes that effectively distinguish B. pseudomallei from its closely related species. Specifically, we selected two specific tags, LC1 and LC2, which we combined with the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR associated proteins (Cas12a) system and recombinase polymerase amplification (RPA) pre-amplification. Our analysis showed that the dual-target RPA-CRISPR/Cas12a assay has a sensitivity of approximately 0.2 copies/reaction and 10 fg genomic DNA for LC1, and 2 copies/reaction and 20 fg genomic DNA for LC2. Additionally, our method can accurately and rapidly detect B. pseudomallei in human blood and moist soil samples using the specific sequence tags mentioned above. In conclusion, the dual-target RPA-CRISPR/Cas12a method is a valuable tool for the rapid and accurate identification of B. pseudomallei in clinical and environmental samples, aiding in the prevention and control of melioidosis.https://www.frontiersin.org/articles/10.3389/fpubh.2023.1153352/fullBurkholderia pseudomalleispecific sequence tagsCRISPR-Cas12avisual detectionspecies discrimination |
| spellingShingle | Jia-Xin Zhang Jian-Hao Xu Bing Yuan Xiao-Dong Wang Xu-hu Mao Jing-Lin Wang Xiang-Li-Lan Zhang Yuan Yuan Detection of Burkholderia pseudomallei with CRISPR-Cas12a based on specific sequence tags Frontiers in Public Health Burkholderia pseudomallei specific sequence tags CRISPR-Cas12a visual detection species discrimination |
| title | Detection of Burkholderia pseudomallei with CRISPR-Cas12a based on specific sequence tags |
| title_full | Detection of Burkholderia pseudomallei with CRISPR-Cas12a based on specific sequence tags |
| title_fullStr | Detection of Burkholderia pseudomallei with CRISPR-Cas12a based on specific sequence tags |
| title_full_unstemmed | Detection of Burkholderia pseudomallei with CRISPR-Cas12a based on specific sequence tags |
| title_short | Detection of Burkholderia pseudomallei with CRISPR-Cas12a based on specific sequence tags |
| title_sort | detection of burkholderia pseudomallei with crispr cas12a based on specific sequence tags |
| topic | Burkholderia pseudomallei specific sequence tags CRISPR-Cas12a visual detection species discrimination |
| url | https://www.frontiersin.org/articles/10.3389/fpubh.2023.1153352/full |
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