CRISPR/Cas12a combined with RPA for detection of T. gondii in mouse whole blood
Abstract Background Toxoplasma gondii is an opportunistic protozoan that is ubiquitous in humans and animals. It can invade any human organ and cause severe diseases, including toxoplasma ophthalmopathy, meningoencephalitis, and liver necrosis. Porcine toxoplasmosis is prevalent in China. CRISPR (Cl...
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| Format: | Article |
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BMC
2023-07-01
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| Series: | Parasites & Vectors |
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| Online Access: | https://doi.org/10.1186/s13071-023-05868-0 |
| _version_ | 1797769643374411776 |
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| author | Xiaofeng Wang Miao Cheng Shuqi Yang Chen Xing Qian Li Yating Zhu Yongsheng Ji Yinan Du |
| author_facet | Xiaofeng Wang Miao Cheng Shuqi Yang Chen Xing Qian Li Yating Zhu Yongsheng Ji Yinan Du |
| author_sort | Xiaofeng Wang |
| collection | DOAJ |
| description | Abstract Background Toxoplasma gondii is an opportunistic protozoan that is ubiquitous in humans and animals. It can invade any human organ and cause severe diseases, including toxoplasma ophthalmopathy, meningoencephalitis, and liver necrosis. Porcine toxoplasmosis is prevalent in China. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and Cas (CRISPR-Associated Protein) systems are widely used for gene editing and pathogen detection. CRISPR-based diagnostics are molecular assays that have been developed to detect parasites with high sensitivity and specificity. Methods This study aimed to establish a combined CRISPR/Cas12a and RPA rapid detection method for T. gondii by targeting the B1 gene and 529 bp repeat element (529 RE). The detection results could be visualized by the fluorescence or lateral flow strips (LFS). The sensitivity and specificity of the method were evaluated, and T. gondii-infected mouse blood was used for detection. Results The results indicated that the established method for T. gondii detection was satisfactory, with a detection limit of 1.5 cp/μl for the two loci. Moreover, the B1 gene could detect 1 tachyzoite per reaction, and the 529 RE could detect 0.1 tachyzoite per reaction, consistently with the highly sensitive nested polymerase chain reaction (PCR) results. The method was suitable for strains, including RH, and did not cross-react with other protozoa DNA with similar habits. The T. gondii-infected mouse blood samples were all positive for T. gondii at 1, 3, and 5 days post infection (dpi). Conclusions This study established a rapid, sensitive, and time-saving DNA detection method for T. gondii that has the potential to be an alternative tool for T. gondii detection in the field. Graphical abstract |
| first_indexed | 2024-03-12T21:12:01Z |
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| id | doaj.art-790e76780d5b4fd3b5360e6fe4c37076 |
| institution | Directory Open Access Journal |
| issn | 1756-3305 |
| language | English |
| last_indexed | 2024-03-12T21:12:01Z |
| publishDate | 2023-07-01 |
| publisher | BMC |
| record_format | Article |
| series | Parasites & Vectors |
| spelling | doaj.art-790e76780d5b4fd3b5360e6fe4c370762023-07-30T11:08:58ZengBMCParasites & Vectors1756-33052023-07-0116111110.1186/s13071-023-05868-0CRISPR/Cas12a combined with RPA for detection of T. gondii in mouse whole bloodXiaofeng Wang0Miao Cheng1Shuqi Yang2Chen Xing3Qian Li4Yating Zhu5Yongsheng Ji6Yinan Du7The Key Laboratory of Microbiology and Parasitology of Anhui Province, the Key Laboratory of Zoonoses of High Institutions in Anhui, Department of Pathogen Biology, School of Basic Medical Sciences, Anhui Medical UniversityThe Key Laboratory of Microbiology and Parasitology of Anhui Province, the Key Laboratory of Zoonoses of High Institutions in Anhui, Department of Pathogen Biology, School of Basic Medical Sciences, Anhui Medical UniversityThe Key Laboratory of Microbiology and Parasitology of Anhui Province, the Key Laboratory of Zoonoses of High Institutions in Anhui, Department of Pathogen Biology, School of Basic Medical Sciences, Anhui Medical UniversityThe Key Laboratory of Microbiology and Parasitology of Anhui Province, the Key Laboratory of Zoonoses of High Institutions in Anhui, Department of Pathogen Biology, School of Basic Medical Sciences, Anhui Medical UniversityThe Key Laboratory of Microbiology and Parasitology of Anhui Province, the Key Laboratory of Zoonoses of High Institutions in Anhui, Department of Pathogen Biology, School of Basic Medical Sciences, Anhui Medical UniversityThe Key Laboratory of Microbiology and Parasitology of Anhui Province, the Key Laboratory of Zoonoses of High Institutions in Anhui, Department of Pathogen Biology, School of Basic Medical Sciences, Anhui Medical UniversitySchool of Basic Medical Sciences, Division of Life Sciences and Medicine, University of Science and Technology of ChinaThe Key Laboratory of Microbiology and Parasitology of Anhui Province, the Key Laboratory of Zoonoses of High Institutions in Anhui, Department of Pathogen Biology, School of Basic Medical Sciences, Anhui Medical UniversityAbstract Background Toxoplasma gondii is an opportunistic protozoan that is ubiquitous in humans and animals. It can invade any human organ and cause severe diseases, including toxoplasma ophthalmopathy, meningoencephalitis, and liver necrosis. Porcine toxoplasmosis is prevalent in China. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) and Cas (CRISPR-Associated Protein) systems are widely used for gene editing and pathogen detection. CRISPR-based diagnostics are molecular assays that have been developed to detect parasites with high sensitivity and specificity. Methods This study aimed to establish a combined CRISPR/Cas12a and RPA rapid detection method for T. gondii by targeting the B1 gene and 529 bp repeat element (529 RE). The detection results could be visualized by the fluorescence or lateral flow strips (LFS). The sensitivity and specificity of the method were evaluated, and T. gondii-infected mouse blood was used for detection. Results The results indicated that the established method for T. gondii detection was satisfactory, with a detection limit of 1.5 cp/μl for the two loci. Moreover, the B1 gene could detect 1 tachyzoite per reaction, and the 529 RE could detect 0.1 tachyzoite per reaction, consistently with the highly sensitive nested polymerase chain reaction (PCR) results. The method was suitable for strains, including RH, and did not cross-react with other protozoa DNA with similar habits. The T. gondii-infected mouse blood samples were all positive for T. gondii at 1, 3, and 5 days post infection (dpi). Conclusions This study established a rapid, sensitive, and time-saving DNA detection method for T. gondii that has the potential to be an alternative tool for T. gondii detection in the field. Graphical abstracthttps://doi.org/10.1186/s13071-023-05868-0T. gondiiCRISPR/Cas12aNucleic acid testingLFSMouse blood |
| spellingShingle | Xiaofeng Wang Miao Cheng Shuqi Yang Chen Xing Qian Li Yating Zhu Yongsheng Ji Yinan Du CRISPR/Cas12a combined with RPA for detection of T. gondii in mouse whole blood Parasites & Vectors T. gondii CRISPR/Cas12a Nucleic acid testing LFS Mouse blood |
| title | CRISPR/Cas12a combined with RPA for detection of T. gondii in mouse whole blood |
| title_full | CRISPR/Cas12a combined with RPA for detection of T. gondii in mouse whole blood |
| title_fullStr | CRISPR/Cas12a combined with RPA for detection of T. gondii in mouse whole blood |
| title_full_unstemmed | CRISPR/Cas12a combined with RPA for detection of T. gondii in mouse whole blood |
| title_short | CRISPR/Cas12a combined with RPA for detection of T. gondii in mouse whole blood |
| title_sort | crispr cas12a combined with rpa for detection of t gondii in mouse whole blood |
| topic | T. gondii CRISPR/Cas12a Nucleic acid testing LFS Mouse blood |
| url | https://doi.org/10.1186/s13071-023-05868-0 |
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