{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,3]],"date-time":"2026-08-03T16:07:18Z","timestamp":1785773238452,"version":"3.56.0"},"reference-count":56,"publisher":"FapUNIFESP (SciELO)","issue":"1","license":[{"start":{"date-parts":[[2022,1,1]],"date-time":"2022-01-01T00:00:00Z","timestamp":1640995200000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2022,1,1]],"date-time":"2022-01-01T00:00:00Z","timestamp":1640995200000},"content-version":"am","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"},{"start":{"date-parts":[[2022,1,1]],"date-time":"2022-01-01T00:00:00Z","timestamp":1640995200000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Rev. Bras. Parasitol. Vet."],"abstract":"<jats:p>Abstract The aim of this study was to validate a one-tube nested real-time PCR assay followed by genetic sequencing to detect and identify Cryptosporidium species and genotypes in birds. A total of 443 genomic DNA extracted from avian fecal samples were analyzed by one-tube nested real-time PCR and conventional nested PCR. By one-tube nested real-time PCR, 90\/443 (20.3%) samples were positive for Cryptosporidium spp. In contrast, 36\/443 (8.1%) samples were positive for Cryptosporidium spp. by conventional nested PCR. The analytical sensitivity test showed that one-tube nested real-time PCR detects approximately 0.5 oocyst (2 sporozoites) per reaction. An evaluation of analytical specificity did not reveal amplification of microorganisms that commonly present nonspecific amplification with primers used for the diagnosis of Cryptosporidium spp. The repeatability analysis showed the same result in 27 out of 30 samples (90%). As for the reproducibility of one-tube nested real-time PCR, 24 of the 30 samples examined (80%) showed the same result. All the 90 samples amplified by one-tube real-time nested PCR were successfully sequenced, leading to the identification of C. baileyi, C. galli, C. meleagridis, C. proventriculi, and Cryptosporidium avian genotype I. Genetic sequencing of conventional nested PCR amplicons was successful in 10\/36 (27.8%) of positive samples.<\/jats:p>","DOI":"10.1590\/s1984-29612022017","type":"journal-article","created":{"date-parts":[[2022,3,22]],"date-time":"2022-03-22T12:25:09Z","timestamp":1647951909000},"source":"Crossref","is-referenced-by-count":7,"title":["Validation of a one-tube nested real-time PCR assay for the detection of Cryptosporidium spp. in avian fecal samples"],"prefix":"10.1590","volume":"31","author":[{"given":"Bruna Nicoleti","family":"Santana","sequence":"first","affiliation":[{"name":"Universidade Estadual Paulista,  Brasil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Elis Domingos","family":"Ferrari","sequence":"additional","affiliation":[{"name":"Universidade Estadual Paulista,  Brasil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Alex Akira","family":"Nakamura","sequence":"additional","affiliation":[{"name":"Universidade Estadual Paulista,  Brasil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Giane Serafim da","family":"Silva","sequence":"additional","affiliation":[{"name":"Instituto Biol\u00f3gico,  Brasil"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0063-5172","authenticated-orcid":false,"given":"Marcelo Vasconcelos","family":"Meireles","sequence":"additional","affiliation":[{"name":"Universidade Estadual Paulista,  Brasil"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"530","published-online":{"date-parts":[[2022]]},"reference":[{"issue":"6","key":"ref1","doi-asserted-by":"crossref","first-page":"1491","DOI":"10.1007\/s00436-010-1810-5","article-title":"Multilocus genotypic analysis of Cryptosporidium isolates from cockatiels, Japan","volume":"106","author":"Abe N","year":"2010","journal-title":"Parasitol Res"},{"issue":"5","key":"ref2","doi-asserted-by":"crossref","first-page":"89S","DOI":"10.1111\/j.1550-7408.1996.tb05015.x","article-title":"Improved purification methods for calf-derived Cryptosporidium parvum oocysts using discontinuous sucrose and cesium chloride gradients","volume":"43","author":"Arrowood MJ","year":"1996","journal-title":"J Eukaryot Microbiol"},{"issue":"2","key":"ref3","doi-asserted-by":"crossref","first-page":"209","DOI":"10.1586\/14737159.5.2.209","article-title":"Basic principles of real-time quantitative PCR","volume":"5","author":"Arya M","year":"2005","journal-title":"Expert Rev Mol Diagn"},{"key":"ref4","doi-asserted-by":"crossref","first-page":"40","DOI":"10.1016\/j.jviromet.2013.10.029","article-title":"Development of a novel single tube nested PCR for enhanced detection of cytomegalovirus DNA from dried blood spots","volume":"196","author":"Atkinson C","year":"2014","journal-title":"J Virol Methods"},{"issue":"2","key":"ref5","doi-asserted-by":"crossref","first-page":"52","DOI":"10.33736\/bjrst.278.2013","article-title":"The prevalence of Cryptosporidium oocysts in birds in Zaria, Nigeria","volume":"2","author":"Bamaiyi PH","year":"2013","journal-title":"Borneo J Resource Sci Technol"},{"issue":"3","key":"ref6","doi-asserted-by":"crossref","first-page":"442","DOI":"10.3382\/ps.0660442","article-title":"Experimental cryptosporidiosis in broiler chickens","volume":"66","author":"Blagburn BL","year":"1987","journal-title":"Poult Sci"},{"issue":"3","key":"ref7","first-page":"155","article-title":"Pitfalls of quantitative real-time reverse-transcription polymerase chain reaction","volume":"15","author":"Bustin SA","year":"2004","journal-title":"J Biomol Tech"},{"issue":"1","key":"ref8","first-page":"61","article-title":"Detection and molecular characterization of Cryptosporidium spp. in captive canaries (Serinus canaria) using different diagnostic methods","volume":"27","author":"Camargo VDS","year":"2018","journal-title":"Rev Bras Parasitol Vet"},{"issue":"4","key":"ref9","doi-asserted-by":"crossref","first-page":"299","DOI":"10.1016\/j.diagmicrobio.2014.08.009","article-title":"Development of a highly sensitive one-tube nested real-time PCR for detecting Mycobacterium tuberculosis.","volume":"80","author":"Choi Y","year":"2014","journal-title":"Diagn Microbiol Infect Dis"},{"issue":"1","key":"ref10","doi-asserted-by":"crossref","first-page":"228","DOI":"10.1016\/j.foodres.2012.12.006","article-title":"Novel approach based on single-tube nested real-time PCR to detect almond allergens in foods","volume":"51","author":"Costa J","year":"2013","journal-title":"Food Res Int"},{"issue":"2","key":"ref11","doi-asserted-by":"crossref","first-page":"289","DOI":"10.1111\/j.1550-7408.1986.tb05608.x","article-title":"The life cycle of Cryptosporidium baileyi n. sp. 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