Development and evaluation of a rapid RPA/CRISPR-based detection of Francisella tularensis
Francisella tularensis is a dangerous pathogen that causes an extremely contagious zoonosis in humans named tularemia. Given its low-dose morbidity, the potential to be fatal, and aerosol spread, it is regarded as a severe threat to public health. The US Centers for Disease Control and Prevention (C...
| Main Authors: | , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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Frontiers Media S.A.
2022-08-01
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| Series: | Frontiers in Microbiology |
| Subjects: | |
| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2022.901520/full |
| _version_ | 1798037776064577536 |
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| author | Jian-Hao Xu Jian-Hao Xu Lin Kang Bing Yuan Zi-Han Feng Shi-Qing Li Jing Wang Ya-Ru Wang Ya-Ru Wang Wen-Wen Xin Shan Gao Jia-Xin Li Yan-Song Sun Jing-Lin Wang Jing-Lin Wang Yuan Yuan |
| author_facet | Jian-Hao Xu Jian-Hao Xu Lin Kang Bing Yuan Zi-Han Feng Shi-Qing Li Jing Wang Ya-Ru Wang Ya-Ru Wang Wen-Wen Xin Shan Gao Jia-Xin Li Yan-Song Sun Jing-Lin Wang Jing-Lin Wang Yuan Yuan |
| author_sort | Jian-Hao Xu |
| collection | DOAJ |
| description | Francisella tularensis is a dangerous pathogen that causes an extremely contagious zoonosis in humans named tularemia. Given its low-dose morbidity, the potential to be fatal, and aerosol spread, it is regarded as a severe threat to public health. The US Centers for Disease Control and Prevention (CDC) has classified it as a category A potential agent for bioterrorism and a Tier 1 Select Agent. Herein, we combined recombinase polymerase amplification (RPA) with CRISPR/Cas12a system to select the F. tularensis target gene (TUL4), creating a two-pronged rapid and ultrasensitive diagnostic method for detecting F. tularensis. The real-time RPA (RT-RPA) assay detected F. tularensis within 10 min at a sensitivity of 5 copies/reaction, F. tularensis genomic DNA of 5 fg, and F. tularensis of 2 × 102 CFU/ml; the RPA-CRISPR/Cas12a assay detects F. tularensis within 40 min at a sensitivity of 0.5 copies/reaction, F. tularensis genomic DNA of 1 fg, and F. tularensis of 2 CFU/ml. Furthermore, the evaluation of specificity showed that both assays were highly specific to F. tularensis. More importantly, in a test of prepared simulated blood and sewage samples, the RT-RPA assay results were consistent with RT-PCR assay results, and the RPA-CRISPR/Cas12a assay could detect a minute amount of F. tularensis genomic DNA (2.5 fg). There was no nonspecific detection with blood samples and sewage samples, giving the tests a high practical application value. For example, in on-site and epidemic areas, the RT-RPA was used for rapid screening and the RPA-CRISPR/Cas12a assay was used for more accurate diagnosis. |
| first_indexed | 2024-04-11T21:31:10Z |
| format | Article |
| id | doaj.art-5f3cb406d690416aa494b2b9e560754e |
| institution | Directory Open Access Journal |
| issn | 1664-302X |
| language | English |
| last_indexed | 2024-04-11T21:31:10Z |
| publishDate | 2022-08-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj.art-5f3cb406d690416aa494b2b9e560754e2022-12-22T04:01:59ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2022-08-011310.3389/fmicb.2022.901520901520Development and evaluation of a rapid RPA/CRISPR-based detection of Francisella tularensisJian-Hao Xu0Jian-Hao Xu1Lin Kang2Bing Yuan3Zi-Han Feng4Shi-Qing Li5Jing Wang6Ya-Ru Wang7Ya-Ru Wang8Wen-Wen Xin9Shan Gao10Jia-Xin Li11Yan-Song Sun12Jing-Lin Wang13Jing-Lin Wang14Yuan Yuan15State Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences (AMMS), Beijing, ChinaSchool of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, ChinaState Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences (AMMS), Beijing, ChinaState Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences (AMMS), Beijing, ChinaDepartment of Disease Control and Prevention, The No. 96609 Hospital of Chinese People's Liberation Army, Yinchuan, ChinaState Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences (AMMS), Beijing, ChinaState Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences (AMMS), Beijing, ChinaState Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences (AMMS), Beijing, ChinaFaculty of Life Science and Technology, Kunming University of Science and Technology, Kunming, ChinaState Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences (AMMS), Beijing, ChinaState Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences (AMMS), Beijing, ChinaState Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences (AMMS), Beijing, ChinaState Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences (AMMS), Beijing, ChinaState Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences (AMMS), Beijing, ChinaSchool of Life Sciences, Fujian Agriculture and Forestry University, Fuzhou, ChinaState Key Laboratory of Pathogen and Biosecurity, Beijing Institute of Microbiology and Epidemiology, Academy of Military Medical Sciences (AMMS), Beijing, ChinaFrancisella tularensis is a dangerous pathogen that causes an extremely contagious zoonosis in humans named tularemia. Given its low-dose morbidity, the potential to be fatal, and aerosol spread, it is regarded as a severe threat to public health. The US Centers for Disease Control and Prevention (CDC) has classified it as a category A potential agent for bioterrorism and a Tier 1 Select Agent. Herein, we combined recombinase polymerase amplification (RPA) with CRISPR/Cas12a system to select the F. tularensis target gene (TUL4), creating a two-pronged rapid and ultrasensitive diagnostic method for detecting F. tularensis. The real-time RPA (RT-RPA) assay detected F. tularensis within 10 min at a sensitivity of 5 copies/reaction, F. tularensis genomic DNA of 5 fg, and F. tularensis of 2 × 102 CFU/ml; the RPA-CRISPR/Cas12a assay detects F. tularensis within 40 min at a sensitivity of 0.5 copies/reaction, F. tularensis genomic DNA of 1 fg, and F. tularensis of 2 CFU/ml. Furthermore, the evaluation of specificity showed that both assays were highly specific to F. tularensis. More importantly, in a test of prepared simulated blood and sewage samples, the RT-RPA assay results were consistent with RT-PCR assay results, and the RPA-CRISPR/Cas12a assay could detect a minute amount of F. tularensis genomic DNA (2.5 fg). There was no nonspecific detection with blood samples and sewage samples, giving the tests a high practical application value. For example, in on-site and epidemic areas, the RT-RPA was used for rapid screening and the RPA-CRISPR/Cas12a assay was used for more accurate diagnosis.https://www.frontiersin.org/articles/10.3389/fmicb.2022.901520/fullFrancisella tularensisrecombinase polymerase amplificationCRISPR/Cas12arapid detectionhigh-sensitive |
| spellingShingle | Jian-Hao Xu Jian-Hao Xu Lin Kang Bing Yuan Zi-Han Feng Shi-Qing Li Jing Wang Ya-Ru Wang Ya-Ru Wang Wen-Wen Xin Shan Gao Jia-Xin Li Yan-Song Sun Jing-Lin Wang Jing-Lin Wang Yuan Yuan Development and evaluation of a rapid RPA/CRISPR-based detection of Francisella tularensis Frontiers in Microbiology Francisella tularensis recombinase polymerase amplification CRISPR/Cas12a rapid detection high-sensitive |
| title | Development and evaluation of a rapid RPA/CRISPR-based detection of Francisella tularensis |
| title_full | Development and evaluation of a rapid RPA/CRISPR-based detection of Francisella tularensis |
| title_fullStr | Development and evaluation of a rapid RPA/CRISPR-based detection of Francisella tularensis |
| title_full_unstemmed | Development and evaluation of a rapid RPA/CRISPR-based detection of Francisella tularensis |
| title_short | Development and evaluation of a rapid RPA/CRISPR-based detection of Francisella tularensis |
| title_sort | development and evaluation of a rapid rpa crispr based detection of francisella tularensis |
| topic | Francisella tularensis recombinase polymerase amplification CRISPR/Cas12a rapid detection high-sensitive |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2022.901520/full |
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