{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,12]],"date-time":"2026-08-12T18:27:50Z","timestamp":1786559270346,"version":"build-2736575974"},"reference-count":25,"publisher":"Public Library of Science (PLoS)","issue":"8","license":[{"start":{"date-parts":[[2026,8,12]],"date-time":"2026-08-12T00:00:00Z","timestamp":1786492800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/publicdomain\/zero\/1.0\/"}],"content-domain":{"domain":["www.plosone.org"],"crossmark-restriction":false},"short-container-title":["PLoS One"],"abstract":"<jats:p>\n                    Increased cases of cyclosporiasis within the United States linked to domestically grown fresh produce highlight the need to identify sources of environmental contamination for\n                    <jats:italic>Cyclospora cayetanensis<\/jats:italic>\n                    . Recently, the Mit1C real-time PCR assay was validated by the U.S. FDA as the US-FDA\u2019s BAM 19c for molecular detection of\n                    <jats:italic>C. cayetanensis<\/jats:italic>\n                    Cox3 gene in agricultural water using the now discontinued ABI-7500 platform. This study optimized the Mit1C real-time PCR assay on the Bio-Rad CFX96 platform for molecular detection of\n                    <jats:italic>C. cayetanensis<\/jats:italic>\n                    in environmental water. Using the Mit1AA synthetic fragment suspended in a 5 \u00b5g\/mL salmon sperm carrier for standard curves, various combinations of cycling times, annealing temperatures, and primer\/probe concentrations were optimized to achieve reproducible, sensitive, specific, and efficient amplification signal. Cross-reactivity was addressed with an exclusivity panel alongside an agricultural water positive control spiked with 200\n                    <jats:italic>C. cayetanensis<\/jats:italic>\n                    oocysts and com-pared on both platforms. Results suggested only decreasing the annealing temperature (66\u00b0C), increasing cycles (42), and increasing cycle cutoff (Cq\u2009\u2264\u200940) to achieve performance comparable to the ABI-7500. Directly utilizing the ABI-7500 protocol for the Bio-Rad CFX96 platform diminished assay sensitivity and efficiency. We adapted the Mit1C real-time PCR protocol for the Bio-Rad CFX96 platform and suggest that instrument differences can have considerable impact on assay performance.\n                  <\/jats:p>","DOI":"10.1371\/journal.pone.0355957","type":"journal-article","created":{"date-parts":[[2026,8,12]],"date-time":"2026-08-12T17:34:13Z","timestamp":1786556053000},"page":"e0355957","update-policy":"https:\/\/doi.org\/10.1371\/journal.pone.corrections_policy","source":"Crossref","is-referenced-by-count":0,"title":["Optimization of the Mit1C real-time PCR assay using Bio-Rad CFX96 for the detection of Cyclospora cayetanensis in agricultural waters"],"prefix":"10.1371","volume":"21","author":[{"given":"Khai","family":"Truong","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Miely","family":"Suarez","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mauricio","family":"Durigan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Laura","family":"Ewing-Peeples","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mark C.","family":"Jenkins","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Aminata","family":"Kilungo","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Margarethe A.","family":"Cooper","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Kerry K.","family":"Cooper","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7898-2129","authenticated-orcid":true,"given":"Gerardo U.","family":"Lopez","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"340","published-online":{"date-parts":[[2026,8,12]]},"reference":[{"issue":"9","key":"pone.0355957.ref001","doi-asserted-by":"crossref","first-page":"2171","DOI":"10.3390\/microorganisms11092171","article-title":"Cyclospora cayetanensis: a perspective (2020\u20132023) with emphasis on epidemiology and detection methods","volume":"11","author":"S Almeria","year":"2023","journal-title":"Microorganisms"},{"issue":"23","key":"pone.0355957.ref002","doi-asserted-by":"crossref","DOI":"10.1128\/AEM.01595-20","article-title":"Dead-End ultrafiltration and DNA-based methods for detection of Cyclospora cayetanensis in agricultural water","volume":"86","author":"M Durigan","year":"2020","journal-title":"Appl Environ Microbiol"},{"key":"pone.0355957.ref003","unstructured":"U.S. Centers for Disease Control and Prevention. Parasites \u2013 Cyclosporiasis (Cyclospora Infection). https:\/\/www.cdc.gov\/parasites\/cyclosporiasis\/outbreaks\/index.html. Accessed 2024 February 5."},{"issue":"1","key":"pone.0355957.ref004","doi-asserted-by":"crossref","first-page":"218","DOI":"10.1128\/CMR.00026-09","article-title":"Update on Cyclospora cayetanensis, a food-borne and waterborne parasite","volume":"23","author":"YR Ortega","year":"2010","journal-title":"Clin Microbiol Rev"},{"issue":"9","key":"pone.0355957.ref005","doi-asserted-by":"crossref","DOI":"10.3390\/microorganisms7090317","article-title":"Cyclospora cayetanensis and cyclosporiasis: an update","volume":"7","author":"S Almeria","year":"2019","journal-title":"Microorganisms"},{"issue":"7","key":"pone.0355957.ref006","doi-asserted-by":"crossref","first-page":"100291","DOI":"10.1016\/j.jfp.2024.100291","article-title":"Detection of cyclospora cayetanensis in food and water samples: optimized protocols for specific and sensitive molecular methods from a regulatory agency perspective","volume":"87","author":"M Durigan","year":"2024","journal-title":"J Food Prot"},{"key":"pone.0355957.ref007","doi-asserted-by":"crossref","first-page":"129","DOI":"10.1016\/j.scitotenv.2014.03.036","article-title":"Occurrence of Cryptosporidium, Giardia, and Cyclospora in influent and effluent water at wastewater treatment plants in Arizona","volume":"484","author":"M Kitajima","year":"2014","journal-title":"Sci Total Environ"},{"key":"pone.0355957.ref008","doi-asserted-by":"crossref","first-page":"318","DOI":"10.1016\/j.watres.2016.05.085","article-title":"Neglected waterborne parasitic protozoa and their detection in water","volume":"101","author":"J Plutzer","year":"2016","journal-title":"Water Research"},{"key":"pone.0355957.ref009","doi-asserted-by":"crossref","first-page":"95","DOI":"10.1016\/j.ijfoodmicro.2015.07.002","article-title":"Molecular detection of Cyclospora in water, soil, vegetables and humans in southern Italy signals a need for improved monitoring by health authorities","volume":"211","author":"A Giangaspero","year":"2015","journal-title":"Int J Food Microbiol"},{"key":"pone.0355957.ref010","article-title":"Food and waterborne protozoan parasites: The African perspective","volume":"20","author":"J Siwila","year":"2020","journal-title":"Food Waterborne Parasitol"},{"issue":"3","key":"pone.0355957.ref011","doi-asserted-by":"crossref","first-page":"117","DOI":"10.2166\/wh.2003.0014","article-title":"Confirmed detection of Cyclospora cayetanesis, Encephalitozoon intestinalis and Cryptosporidium parvum in water used for drinking","volume":"1","author":"SE Dowd","year":"2003","journal-title":"J Water Health"},{"issue":"1","key":"pone.0355957.ref012","doi-asserted-by":"crossref","first-page":"210","DOI":"10.2166\/wh.2014.093","article-title":"Cyclospora cayetanensis travels in tap water on Italian trains","volume":"13","author":"A Giangaspero","year":"2015","journal-title":"J Water Health"},{"key":"pone.0355957.ref013","article-title":"The prevalence of Cyclospora cayetanensis in water: a systematic review and meta-analysis","volume":"150","author":"T Naganathan","year":"2021","journal-title":"Epidemiol Infect"},{"key":"pone.0355957.ref014","unstructured":"U.S. Food and Drug Administration. BAM 19c: Dead-end ultrafiltration for the detection of Cyclospora cayetanensis from agricultural water. 2024. https:\/\/www.fda.gov\/food\/laboratory-methods-food\/bam-19c-dead-end-ultrafiltration-detection-Cyclospora-cayetanensis-agricultural-water"},{"key":"pone.0355957.ref015","doi-asserted-by":"crossref","first-page":"170","DOI":"10.1016\/j.fm.2017.08.008","article-title":"Interlaboratory validation of an improved method for detection of Cyclospora cayetanensis in produce using a real-time PCR assay","volume":"69","author":"HR Murphy","year":"2018","journal-title":"Food Microbiol"},{"issue":"2","key":"pone.0355957.ref016","doi-asserted-by":"crossref","first-page":"100037","DOI":"10.1016\/j.jfp.2022.100037","article-title":"Development and single laboratory evaluation of a refined and specific real-time PCR Detection Method, Using Mitochondrial Primers (Mit1C), for the detection of cyclospora cayetanensis in produce","volume":"86","author":"KV Balan","year":"2023","journal-title":"J Food Prot"},{"key":"pone.0355957.ref017","doi-asserted-by":"crossref","first-page":"1939","DOI":"10.3389\/fmicb.2019.01939","article-title":"A new protocol for molecular detection of Cyclospora cayetanensis as contaminants of berry fruits","volume":"10","author":"TT Temesgen","year":"2019","journal-title":"Front Microbiol"},{"issue":"3","key":"pone.0355957.ref018","doi-asserted-by":"crossref","first-page":"559","DOI":"10.3390\/microorganisms10030559","article-title":"Verification and use of the US-FDA BAM 19b method for detection of Cyclospora cayetanensis in a survey of fresh produce by CFIA laboratory","volume":"10","author":"L Lalonde","year":"2022","journal-title":"Microorganisms"},{"key":"pone.0355957.ref019","unstructured":"U.S. Food and Drug Administration. Other analytical methods of interest to the foods program. https:\/\/www.fda.gov\/food\/laboratory-methods-food\/other-analytical-methods-interest-foods-program. 2024. Accessed 2025 July 21."},{"issue":"4","key":"pone.0355957.ref020","doi-asserted-by":"crossref","first-page":"366","DOI":"10.1111\/j.1472-765X.2010.02804.x","article-title":"A versatile internal control for use as DNA in real-time PCR and as RNA in real-time reverse transcription PCR assays","volume":"50","author":"DM Deer","year":"2010","journal-title":"Lett Appl Microbiol"},{"issue":"7","key":"pone.0355957.ref021","doi-asserted-by":"crossref","first-page":"100309","DOI":"10.1016\/j.jfp.2024.100309","article-title":"Sources and prevalence of Cyclospora cayetanensis in Southeastern U.S. Growing Environments","volume":"87","author":"AM Kahler","year":"2024","journal-title":"J Food Prot"},{"issue":"2","key":"pone.0355957.ref022","doi-asserted-by":"crossref","first-page":"115","DOI":"10.1016\/j.tifs.2014.03.008","article-title":"Guidelines for validation of qualitative real-time PCR methods","volume":"37","author":"S Broeders","year":"2014","journal-title":"Trends in Food Science & Technology"},{"issue":"2","key":"pone.0355957.ref023","doi-asserted-by":"crossref","first-page":"75","DOI":"10.1007\/s00769-015-1112-9","article-title":"Validation of qualitative PCR methods on the basis of mathematical\u2013statistical modelling of the probability of detection","volume":"20","author":"S Uhlig","year":"2015","journal-title":"Accred Qual Assur"},{"issue":"9","key":"pone.0355957.ref024","doi-asserted-by":"crossref","first-page":"1762","DOI":"10.3390\/microorganisms10091762","article-title":"Development of a molecular marker based on the mitochondrial genome for detection of Cyclospora cayetanensis in food and water samples","volume":"10","author":"M Durigan","year":"2022","journal-title":"Microorganisms"},{"key":"pone.0355957.ref025","doi-asserted-by":"crossref","DOI":"10.1016\/j.fawpar.2021.e00110","article-title":"Detection of Cyclospora cayetanensis in produce irrigation and wash water using large-volume sampling techniques","volume":"22","author":"AM Kahler","year":"2021","journal-title":"Food and Waterborne Parasitology"}],"container-title":["PLOS One"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0355957","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,8,12]],"date-time":"2026-08-12T17:34:18Z","timestamp":1786556058000},"score":1,"resource":{"primary":{"URL":"https:\/\/dx.plos.org\/10.1371\/journal.pone.0355957"}},"subtitle":[],"editor":[{"given":"Antonio","family":"Pe\u00f1a-Fern\u00e1ndez","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"editor"}]}],"short-title":[],"issued":{"date-parts":[[2026,8,12]]},"references-count":25,"journal-issue":{"issue":"8","published-online":{"date-parts":[[2026,8,12]]}},"URL":"https:\/\/doi.org\/10.1371\/journal.pone.0355957","relation":{},"ISSN":["1932-6203"],"issn-type":[{"value":"1932-6203","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,12]]}}}