{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,6]],"date-time":"2026-07-06T23:30:14Z","timestamp":1783380614810,"version":"3.54.6"},"reference-count":50,"publisher":"MDPI AG","issue":"8","license":[{"start":{"date-parts":[[2021,7,31]],"date-time":"2021-07-31T00:00:00Z","timestamp":1627689600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001809","name":"National Natural Science Foundation of China","doi-asserted-by":"publisher","award":["32002306"],"award-info":[{"award-number":["32002306"]}],"id":[{"id":"10.13039\/501100001809","id-type":"DOI","asserted-by":"publisher"}]},{"name":"the Agricultural Science and Technology Innovation Program (ASTIP)","award":["CAAS-ASTIP-2016-LVRI-03"],"award-info":[{"award-number":["CAAS-ASTIP-2016-LVRI-03"]}]},{"name":"the Yunnan Expert Workstation","award":["202005AF150041"],"award-info":[{"award-number":["202005AF150041"]}]},{"name":"the Veterinary Public Health Innovation Team of Yunnan Province","award":["Not applicable"],"award-info":[{"award-number":["Not applicable"]}]},{"name":"the Start-Up Fund of Shanxi Agricultural University","award":["Not applicable"],"award-info":[{"award-number":["Not applicable"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Microorganisms"],"abstract":"<jats:p>Toxoplasmosis, caused by the intracellular protozoon Toxoplasma gondii, is a significant parasitic zoonosis with a world-wide distribution. As a main transmission route, human infection can be acquired by the ingestion of T. gondii oocysts from the environment (e.g., soil, water, fruits and vegetables). Regarding the detection of T. gondii oocysts in environmental samples, the development of a time-saving, cost-effective and highly sensitive technique is crucial for the surveillance, prevention and control of toxoplasmosis. In this study, we developed a new method by combining recombinase-aided amplification (RAA) with CRISPR-Cas12a, designated as the RAA-Cas12a-Tg system. Here, we compared this system targeting the 529 bp repeat element (529 bp-RE) with the routine PCR targeting both 529 bp-RE and ITS-1 gene, respectively, to assess its ability to detect T. gondii oocysts in soil samples. Our results indicated that the 529 bp RE-based RAA-Cas12a-Tg system was able to detect T. gondii successfully in nearly an hour at body temperature and was more sensitive than the routine PCR assay. The sensitivity of this system reached as low as 1 fM with high specificity. Thus, RAA-Cas12a-Tg system provided a rapid, sensitive and easily operable method for point-of-care detection of T. gondii oocysts in soil, which will facilitate the control of T. gondii infection in humans and animals.<\/jats:p>","DOI":"10.3390\/microorganisms9081644","type":"journal-article","created":{"date-parts":[[2021,8,1]],"date-time":"2021-08-01T21:44:32Z","timestamp":1627854272000},"page":"1644","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":47,"title":["RAA-Cas12a-Tg: A Nucleic Acid Detection System for Toxoplasma gondii Based on CRISPR-Cas12a Combined with Recombinase-Aided Amplification (RAA)"],"prefix":"10.3390","volume":"9","author":[{"given":"Qiao-Ni","family":"Ma","sequence":"first","affiliation":[{"name":"State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Meng","family":"Wang","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0444-8163","authenticated-orcid":false,"given":"Lai-Bao","family":"Zheng","sequence":"additional","affiliation":[{"name":"Wenzhou Key Laboratory of Sanitary Microbiology, Key Laboratory of Laboratory Medicine, Ministry of Education, School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou 325035, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zi-Qin","family":"Lin","sequence":"additional","affiliation":[{"name":"Wenzhou Key Laboratory of Sanitary Microbiology, Key Laboratory of Laboratory Medicine, Ministry of Education, School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou 325035, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5314-5869","authenticated-orcid":false,"given":"Muhammad","family":"Ehsan","sequence":"additional","affiliation":[{"name":"Department of Parasitology, Faculty of Veterinary and Animal Sciences, The Islamia University of Bahawalpur, Bahawalpur 63100, Pakistan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xing-Xing","family":"Xiao","sequence":"additional","affiliation":[{"name":"Wenzhou Key Laboratory of Sanitary Microbiology, Key Laboratory of Laboratory Medicine, Ministry of Education, School of Laboratory Medicine and Life Sciences, Wenzhou Medical University, Wenzhou 325035, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2530-4628","authenticated-orcid":false,"given":"Xing-Quan","family":"Zhu","sequence":"additional","affiliation":[{"name":"State Key Laboratory of Veterinary Etiological Biology, Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China"},{"name":"College of Veterinary Medicine, Shanxi Agricultural University, Taigu, Jinzhong 030801, China"},{"name":"Key Laboratory of Veterinary Public Health of Yunnan Province, College of Veterinary Medicine, Yunnan Agricultural University, Kunming 650201, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2021,7,31]]},"reference":[{"key":"ref_1","unstructured":"Dubey, J.P. (2010). Toxoplasmosis of Animals and Humans, CRC Press. [2nd ed.]."},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"165","DOI":"10.1186\/1756-3305-4-165","article-title":"Toxoplasma gondii infection in humans in China","volume":"4","author":"Zhou","year":"2011","journal-title":"Parasit. Vectors"},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"554","DOI":"10.1086\/590149","article-title":"Management of Toxoplasma gondii infection during pregnancy","volume":"47","author":"Montoya","year":"2008","journal-title":"Clin. Infect. Dis."},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"e146","DOI":"10.1016\/S2352-3018(17)30003-6","article-title":"Toxoplasma gondii and HIV: A never-ending story","volume":"4","author":"Nissapatorn","year":"2017","journal-title":"Lancet HIV"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"109370","DOI":"10.1016\/j.vetpar.2021.109370","article-title":"Global prevalence of Toxoplasma gondii infection in the aborted fetuses and ruminants that had an abortion: A systematic review and meta-analysis","volume":"290","author":"Nayeri","year":"2021","journal-title":"Vet. Parasitol."},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"207","DOI":"10.1016\/j.ijfoodmicro.2004.12.032","article-title":"Foodborne protozoan parasites","volume":"103","author":"Dawson","year":"2005","journal-title":"Int. J. Food Microbiol."},{"key":"ref_7","doi-asserted-by":"crossref","first-page":"357","DOI":"10.1016\/j.ijpara.2017.01.004","article-title":"Spatial distribution of soil contaminated with Toxoplasma gondii oocysts in relation to the distribution and use of domestic cat defecation sites on dairy farms","volume":"47","author":"Simon","year":"2017","journal-title":"Int. J. Parasitol."},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"1","DOI":"10.1038\/s41598-019-54073-6","article-title":"First molecular detection of Toxoplasma gondii in vegetable samples in China using qualitative, quantitative real-time PCR and multilocus genotyping","volume":"9","author":"Lass","year":"2019","journal-title":"Sci. Rep."},{"key":"ref_9","doi-asserted-by":"crossref","first-page":"109999","DOI":"10.1016\/j.ecoenv.2019.109999","article-title":"Prevalence, risk factors and genotype distribution of Toxoplasma gondii DNA in soil in China","volume":"189","author":"Cong","year":"2020","journal-title":"Ecotoxicol. Environ. Saf."},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"380","DOI":"10.1186\/s13071-020-04255-3","article-title":"Detection of human intestinal protozoan parasites in vegetables and fruits: A review","volume":"13","author":"Li","year":"2020","journal-title":"Parasit. Vectors"},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"180","DOI":"10.1186\/s13071-020-04040-2","article-title":"Detection of Toxoplasma gondii oocysts in fresh vegetables and berry fruits","volume":"13","author":"Marques","year":"2020","journal-title":"Parasit. Vectors"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"112048","DOI":"10.1016\/j.ecoenv.2021.112048","article-title":"Prevalence, genotypes and risk factors for Toxoplasma gondii contamination in marine bivalve shellfish in offshore waters in eastern China","volume":"213","author":"Cong","year":"2021","journal-title":"Ecotoxicol. Environ. Saf."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"143232","DOI":"10.1016\/j.scitotenv.2020.143232","article-title":"Digging into Toxoplasma gondii infections via soil: A quantitative microbial risk assessment approach","volume":"755","author":"Deng","year":"2021","journal-title":"Sci. Total Environ."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"14","DOI":"10.1051\/parasite\/2018009","article-title":"Assessing viability and infectivity of foodborne and waterborne stages (cysts\/oocysts) of Giardia duodenalis, Cryptosporidium spp., and Toxoplasma gondii: A review of methods","volume":"25","author":"Rousseau","year":"2018","journal-title":"Parasite"},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"292","DOI":"10.1186\/s13071-015-0902-6","article-title":"Diagnosis of toxoplasmosis and typing of Toxoplasma gondii","volume":"8","author":"Liu","year":"2015","journal-title":"Parasit. Vectors"},{"key":"ref_16","first-page":"e00115","article-title":"Epidemiology, pathophysiology, diagnosis, and management of cerebral toxoplasmosis","volume":"34","author":"Elsheikha","year":"2021","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"170","DOI":"10.1111\/j.1469-0691.2004.01073.x","article-title":"Recent trends in molecular diagnostics for Toxoplasma gondii infections","volume":"11","author":"Switaj","year":"2005","journal-title":"Clin. Microbiol. Infect."},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"69","DOI":"10.1016\/S0020-7519(99)00170-8","article-title":"Identification of a 200- to 300-fold repetitive 529 bp DNA fragment in Toxoplasma gondii, and its use for diagnostic and quantitative PCR","volume":"30","author":"Homan","year":"2000","journal-title":"Int. J. Parasitol."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"103938","DOI":"10.1016\/j.micpath.2019.103938","article-title":"Comparison of the RE-529 sequence and B1 gene for Toxoplasma gondii detection in blood samples of the at-risk seropositive cases using uracil DNA glycosylase supplemented loop-mediated isothermal amplification (UDG-LAMP) assay","volume":"140","author":"Valian","year":"2020","journal-title":"Microb. Pathog."},{"key":"ref_20","first-page":"75","article-title":"Comparison between two real-time PCR assays and a nested-PCR for the detection of Toxoplasma gondii","volume":"77","author":"Calderaro","year":"2006","journal-title":"Acta Bio-Medica Atenei Parm."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"2065","DOI":"10.1128\/JCM.39.6.2065-2071.2001","article-title":"Development of a real-time PCR assay for detection of Toxoplasma gondii in pig and mouse tissues","volume":"39","author":"Jauregui","year":"2001","journal-title":"J. Clin. Microbiol."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"296","DOI":"10.1016\/j.vetpar.2011.10.016","article-title":"Comparison of loop-mediated isothermal amplification (LAMP) and real-time PCR method targeting a 529-bp repeat element for diagnosis of toxoplasmosis","volume":"185","author":"Lin","year":"2012","journal-title":"Vet. Parasitol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"573","DOI":"10.3347\/kjp.2013.51.5.573","article-title":"Detection of acute toxoplasmosis in pigs using loop-mediated isothermal amplification and quantitative PCR","volume":"51","author":"Wang","year":"2013","journal-title":"Korean J. Parasitol."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"32","DOI":"10.1186\/s13073-018-0543-4","article-title":"Pathogen detection in the CRISPR\u2013Cas era","volume":"10","author":"Sashital","year":"2018","journal-title":"Genome Med."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"01307","DOI":"10.1128\/JCM.01307-18","article-title":"CRISPR-Cas biology and its application to infectious diseases","volume":"57","author":"Strich","year":"2019","journal-title":"J. Clin. Microbiol."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"439","DOI":"10.1126\/science.aaq0179","article-title":"Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6","volume":"360","author":"Gootenberg","year":"2018","journal-title":"Science"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"438","DOI":"10.1126\/science.aam9321","article-title":"Nucleic acid detection with CRISPR-Cas13a\/C2c2","volume":"356","author":"Gootenberg","year":"2017","journal-title":"Science"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"839","DOI":"10.1126\/science.aav4294","article-title":"Programmed DNA destruction by miniature CRISPR-Cas14 enzymes","volume":"362","author":"Harrington","year":"2018","journal-title":"Science"},{"key":"ref_29","doi-asserted-by":"crossref","first-page":"2830","DOI":"10.3389\/fmicb.2019.02830","article-title":"Cas12a-based on-site and rapid nucleic acid detection of African swine fever","volume":"10","author":"Bai","year":"2019","journal-title":"Front. Microbiol."},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"14846","DOI":"10.1021\/acs.analchem.0c03883","article-title":"Reagents-loaded, automated assay that integrates recombinase-aided amplification and Cas12a nucleic acid detection for a point-of-care test","volume":"92","author":"Chen","year":"2020","journal-title":"Anal. Chem."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"113073","DOI":"10.1016\/j.bios.2021.113073","article-title":"An ultrasensitive CRISPR\/Cas12a based electrochemical biosensor for Listeria monocytogenes detection","volume":"179","author":"Li","year":"2021","journal-title":"Biosens. Bioelectron."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"870","DOI":"10.1038\/s41587-020-0513-4","article-title":"CRISPR-Cas12-based detection of SARS-CoV-2","volume":"38","author":"Broughton","year":"2020","journal-title":"Nat. Biotechnol."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"112068","DOI":"10.1016\/j.bios.2020.112068","article-title":"High-throughput and all-solution phase African swine fever virus (ASFV) detection using CRISPR-Cas12a and fluorescence based point-of-care system","volume":"154","author":"He","year":"2020","journal-title":"Biosens. Bioelectron."},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Xiong, D., Dai, W., Gong, J., Li, G., Liu, N., Wu, W., Pan, J., Chen, C., Jiao, Y., and Deng, H. (2020). Rapid detection of SARS-CoV-2 with CRISPR-Cas12a. PLoS. Biol., 18.","DOI":"10.1371\/journal.pbio.3000978"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"1361","DOI":"10.1080\/22221751.2019.1664939","article-title":"CRISPR-based rapid and ultra-sensitive diagnostic test for Mycobacterium tuberculosis","volume":"8","author":"Ai","year":"2019","journal-title":"Emerg. Microbes Infect."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"1427","DOI":"10.1021\/acssensors.0c00320","article-title":"A one-pot toolbox based on Cas12a\/crRNA enables rapid foodborne pathogen detection at attomolar level","volume":"5","author":"Wang","year":"2020","journal-title":"ACS Sens."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"225","DOI":"10.1016\/j.aca.2020.06.041","article-title":"CRISPR-Cas13a based bacterial detection platform: Sensing pathogen Staphylococcus aureus in food samples","volume":"1127","author":"Zhou","year":"2020","journal-title":"Anal. Chim. Acta"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"25722","DOI":"10.1073\/pnas.2010196117","article-title":"Ultrasensitive CRISPR-based diagnostic for field-applicable detection of Plasmodium species in symptomatic and asymptomatic malaria","volume":"117","author":"Lee","year":"2020","journal-title":"Proc. Natl. Acad. Sci. USA"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"208","DOI":"10.1186\/s13071-021-04709-2","article-title":"CRISPR\/Cas12a-based on-site diagnostics of Cryptosporidium parvum IId-subtype-family from human and cattle fecal samples","volume":"14","author":"Yu","year":"2021","journal-title":"Parasit. Vectors"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"270","DOI":"10.1089\/152791600459894","article-title":"Primer Premier 5","volume":"1","author":"Lalitha","year":"2000","journal-title":"Biotech Softw. Internet Rep."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"492","DOI":"10.1016\/j.isci.2019.08.005","article-title":"Conformational dynamics and cleavage sites of Cas12a are modulated by complementarity between crRNA and DNA","volume":"19","author":"Zhang","year":"2019","journal-title":"iScience"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"4035","DOI":"10.1128\/AEM.70.7.4035-4039.2004","article-title":"Evaluation of a strategy for Toxoplasma gondii oocyst detection in water","volume":"70","author":"Villena","year":"2004","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"91","DOI":"10.1016\/j.ijpara.2013.10.001","article-title":"Toxoplasma gondii within skeletal muscle cells: A critical interplay for food-borne parasite transmission","volume":"44","author":"Swierzy","year":"2014","journal-title":"Int. J. Parasitol."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"228","DOI":"10.1016\/j.watres.2017.02.044","article-title":"Lectin-magnetic separation (LMS) for isolation of Toxoplasma gondii oocysts from concentrated water samples prior to detection by microscopy or qPCR","volume":"114","author":"Harito","year":"2017","journal-title":"Water Res."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"e01189-19","DOI":"10.1128\/AEM.01189-19","article-title":"Toxoplasma gondii oocyst infectivity assessed using a sporocyst-based cell culture assay combined with quantitative PCR for environmental applications","volume":"85","author":"Rousseau","year":"2019","journal-title":"Appl. Environ. Microbiol."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"111632","DOI":"10.1016\/j.bios.2019.111632","article-title":"Sunlight based handheld smartphone spectrometer","volume":"143","author":"Jian","year":"2019","journal-title":"Biosens. Bioelectron."},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"82","DOI":"10.1089\/crispr.2020.0086","article-title":"Development of a Cas12a-based genome editing tool for moderate thermophiles","volume":"4","author":"Mohanraju","year":"2021","journal-title":"CRISPR J."},{"key":"ref_48","doi-asserted-by":"crossref","first-page":"4711","DOI":"10.1038\/s41467-020-18575-6","article-title":"Ultrasensitive and visual detection of SARS-CoV-2 using all-in-one dual CRISPR-Cas12a assay","volume":"11","author":"Ding","year":"2020","journal-title":"Nat. Commun."},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"eabc7802","DOI":"10.1126\/sciadv.abc7802","article-title":"A CRISPR-Cas autocatalysis-driven feedback amplification network for supersensitive DNA diagnostics","volume":"7","author":"Shi","year":"2021","journal-title":"Sci. Adv."},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"1499","DOI":"10.1042\/BST20190564","article-title":"Making the cut(s): How Cas12a cleaves target and non-target DNA","volume":"47","author":"Swarts","year":"2019","journal-title":"Biochem. Soc. Trans."}],"container-title":["Microorganisms"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-2607\/9\/8\/1644\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,10,11]],"date-time":"2025-10-11T06:38:39Z","timestamp":1760164719000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-2607\/9\/8\/1644"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2021,7,31]]},"references-count":50,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2021,8]]}},"alternative-id":["microorganisms9081644"],"URL":"https:\/\/doi.org\/10.3390\/microorganisms9081644","relation":{},"ISSN":["2076-2607"],"issn-type":[{"value":"2076-2607","type":"electronic"}],"subject":[],"published":{"date-parts":[[2021,7,31]]}}}