Rapid and sensitive detection of bovine Theileria annulata parasite based on ERA-CRISPR/Cas12a technology
Theileria annulata, a globally significant blood parasite in livestock, causes substantial economic losses in resource-limited regions by compromising animal health and hindering the development of the livestock industry. To address this, a rapid, reliable, and sensitive diagnostic assay integrating...
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Frontiers Media S.A.
2025-08-01
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| Series: | Frontiers in Microbiology |
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| Online Access: | https://www.frontiersin.org/articles/10.3389/fmicb.2025.1647929/full |
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| author | Xiujuan Feng Yongchang Li Shiyun Li Iqra zafar Mohamed Abdo Rizk Xianyue Fu Zeyun Cui Wei Zhang Yang Zhang Ercha Hu Qingyong Guo Bayin Chahan |
| author_facet | Xiujuan Feng Yongchang Li Shiyun Li Iqra zafar Mohamed Abdo Rizk Xianyue Fu Zeyun Cui Wei Zhang Yang Zhang Ercha Hu Qingyong Guo Bayin Chahan |
| author_sort | Xiujuan Feng |
| collection | DOAJ |
| description | Theileria annulata, a globally significant blood parasite in livestock, causes substantial economic losses in resource-limited regions by compromising animal health and hindering the development of the livestock industry. To address this, a rapid, reliable, and sensitive diagnostic assay integrating enzymatic recombinase amplification (ERA) with CRISPR/Cas12a technology was developed. This assay enables visual interpretation through multiple detection modalities, including UV and blue light illumination. Among three primer pairs and two CRISPR RNA (crRNA) candidates screened, the F3/R3 primer set combined with crRNA1 demonstrated the best performance. The optimized ERA protocol achieved complete amplification within 20 min at 37°C. This assay exhibited high specificity for T. annulata detection, with a sensitivity limit of 10 copies/μL, a 100-fold greater sensitivity than conventional PCR, while completing detection within 40 min. Validation of 51 bovine blood samples from a farm in Turpan, Xinjiang, revealed that PCR detected 12 positive cases (23.5% prevalence), whereas the ERA-CRISPR/Cas12a system identified 15 positive cases (29.4% prevalence). The enhanced detection capability of this integrated method provides crucial technical support for field applications in resource-limited settings, effectively addressing the urgent need for rapid and accurate diagnosis of bovine theileriosis. |
| format | Article |
| id | doaj-art-8e5ace19ae334c65ada23f80d76639f3 |
| institution | Kabale University |
| issn | 1664-302X |
| language | English |
| publishDate | 2025-08-01 |
| publisher | Frontiers Media S.A. |
| record_format | Article |
| series | Frontiers in Microbiology |
| spelling | doaj-art-8e5ace19ae334c65ada23f80d76639f32025-08-20T03:57:36ZengFrontiers Media S.A.Frontiers in Microbiology1664-302X2025-08-011610.3389/fmicb.2025.16479291647929Rapid and sensitive detection of bovine Theileria annulata parasite based on ERA-CRISPR/Cas12a technologyXiujuan Feng0Yongchang Li1Shiyun Li2Iqra zafar3Mohamed Abdo Rizk4Xianyue Fu5Zeyun Cui6Wei Zhang7Yang Zhang8Ercha Hu9Qingyong Guo10Bayin Chahan11Parasitology Laboratory, Veterinary College, Xinjiang Agricultural University, Ürümqi, ChinaParasitology Laboratory, Veterinary College, Xinjiang Agricultural University, Ürümqi, ChinaParasitology Laboratory, Veterinary College, Xinjiang Agricultural University, Ürümqi, ChinaLaboratory of Sustainable Animal Environment, Graduate School of Agriculture Science, Tohoku University, Sendai, JapanDepartment of Internal Medicine and Infectious Diseases, Faculty of Veterinary Medicine, Mansoura University, Mansoura, EgyptParasitology Laboratory, Veterinary College, Xinjiang Agricultural University, Ürümqi, ChinaParasitology Laboratory, Veterinary College, Xinjiang Agricultural University, Ürümqi, ChinaParasitology Laboratory, Veterinary College, Xinjiang Agricultural University, Ürümqi, ChinaParasitology Laboratory, Veterinary College, Xinjiang Agricultural University, Ürümqi, ChinaParasitology Laboratory, Veterinary College, Xinjiang Agricultural University, Ürümqi, ChinaParasitology Laboratory, Veterinary College, Xinjiang Agricultural University, Ürümqi, ChinaParasitology Laboratory, Veterinary College, Xinjiang Agricultural University, Ürümqi, ChinaTheileria annulata, a globally significant blood parasite in livestock, causes substantial economic losses in resource-limited regions by compromising animal health and hindering the development of the livestock industry. To address this, a rapid, reliable, and sensitive diagnostic assay integrating enzymatic recombinase amplification (ERA) with CRISPR/Cas12a technology was developed. This assay enables visual interpretation through multiple detection modalities, including UV and blue light illumination. Among three primer pairs and two CRISPR RNA (crRNA) candidates screened, the F3/R3 primer set combined with crRNA1 demonstrated the best performance. The optimized ERA protocol achieved complete amplification within 20 min at 37°C. This assay exhibited high specificity for T. annulata detection, with a sensitivity limit of 10 copies/μL, a 100-fold greater sensitivity than conventional PCR, while completing detection within 40 min. Validation of 51 bovine blood samples from a farm in Turpan, Xinjiang, revealed that PCR detected 12 positive cases (23.5% prevalence), whereas the ERA-CRISPR/Cas12a system identified 15 positive cases (29.4% prevalence). The enhanced detection capability of this integrated method provides crucial technical support for field applications in resource-limited settings, effectively addressing the urgent need for rapid and accurate diagnosis of bovine theileriosis.https://www.frontiersin.org/articles/10.3389/fmicb.2025.1647929/fullenzymatic recombinase amplificationTheileria annulataCRISPR Cas12aenolasemolecular diagnostics |
| spellingShingle | Xiujuan Feng Yongchang Li Shiyun Li Iqra zafar Mohamed Abdo Rizk Xianyue Fu Zeyun Cui Wei Zhang Yang Zhang Ercha Hu Qingyong Guo Bayin Chahan Rapid and sensitive detection of bovine Theileria annulata parasite based on ERA-CRISPR/Cas12a technology Frontiers in Microbiology enzymatic recombinase amplification Theileria annulata CRISPR Cas12a enolase molecular diagnostics |
| title | Rapid and sensitive detection of bovine Theileria annulata parasite based on ERA-CRISPR/Cas12a technology |
| title_full | Rapid and sensitive detection of bovine Theileria annulata parasite based on ERA-CRISPR/Cas12a technology |
| title_fullStr | Rapid and sensitive detection of bovine Theileria annulata parasite based on ERA-CRISPR/Cas12a technology |
| title_full_unstemmed | Rapid and sensitive detection of bovine Theileria annulata parasite based on ERA-CRISPR/Cas12a technology |
| title_short | Rapid and sensitive detection of bovine Theileria annulata parasite based on ERA-CRISPR/Cas12a technology |
| title_sort | rapid and sensitive detection of bovine theileria annulata parasite based on era crispr cas12a technology |
| topic | enzymatic recombinase amplification Theileria annulata CRISPR Cas12a enolase molecular diagnostics |
| url | https://www.frontiersin.org/articles/10.3389/fmicb.2025.1647929/full |
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