{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,7,13]],"date-time":"2026-07-13T11:06:16Z","timestamp":1783940776173,"version":"3.55.0"},"reference-count":30,"publisher":"MDPI AG","issue":"7","license":[{"start":{"date-parts":[[2026,7,11]],"date-time":"2026-07-11T00:00:00Z","timestamp":1783728000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["CIMB"],"abstract":"<jats:p>Feline feces represent a difficult matrix for molecular diagnostics because stool-derived inhibitors and variable sample composition may compromise DNA recovery and downstream PCR amplification. This paired methodological study evaluated whether a pre-analytical chloroform:methanol cleanup step improves DNA extract quality, post-extraction amplifiability, and molecular detection of selected intestinal protozoa in cats. A total of 105 feline fecal samples were divided into paired aliquots and subjected either to direct automated DNA extraction or to extraction preceded by chloroform:methanol cleanup. DNA concentration and purity were assessed spectrophotometrically using DNA yield, A260\/280, and A260\/230 ratios. Extracts from both workflows were analyzed by PCR for Tritrichomonas foetus, Cryptosporidium spp., Pentatrichomonas hominis, and Giardia duodenalis. In addition, a post-extraction spike-recovery experiment was performed on selected paired extracts to directly evaluate PCR inhibition. Cleanup significantly increased DNA concentration and improved A260\/280 and A260\/230 ratios. It was also associated with additional Cryptosporidium and Giardia detections and significantly lower Cryptosporidium Ct values among paired positives. Spike-recovery testing showed that direct extracts markedly delayed amplification of an exogenous Cryptosporidium meleagridis 18S spike, whereas cleanup-based extracts showed significantly lower Ct delay and higher spike recovery. All Cryptosporidium-positive sequences were identified as Cryptosporidium felis. Sequencing of the six Giardia beta-giardin amplicons revealed four sequences assigned to assemblage F, one sequence assigned to assemblage B, and one sequence assigned to assemblage D. These findings support chloroform:methanol cleanup as a useful pre-analytical strategy for improving feline fecal DNA extract quality and reducing post-extraction PCR inhibition, although broader validation across additional extraction platforms and larger positive sample sets is required.<\/jats:p>","DOI":"10.3390\/cimb48070707","type":"journal-article","created":{"date-parts":[[2026,7,13]],"date-time":"2026-07-13T09:51:09Z","timestamp":1783936269000},"page":"707","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Pre-Analytical Cleanup of Feline Feces Improves DNA Extract Quality, Reduces Post-Extraction PCR Inhibition, and Enhances Molecular Detectability of Intestinal Protozoa"],"prefix":"10.3390","volume":"48","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0845-0810","authenticated-orcid":false,"given":"Dawid","family":"Ja\u0144czak","sequence":"first","affiliation":[{"name":"Department of Infectious and Invasive Diseases and Veterinary Administration, Institute of Veterinary Medicine, Faculty of Biological and Veterinary Sciences, Nicolaus Copernicus University, Lwowska 1, 87-100 Toru\u0144, Poland"},{"name":"Animallab Veterinary Laboratory, \u015arodkowa 2\/4, 03-340 Warsaw, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0004-2601-3106","authenticated-orcid":false,"given":"Aleksandra Kornelia","family":"Maj","sequence":"additional","affiliation":[{"name":"Animallab Veterinary Laboratory, \u015arodkowa 2\/4, 03-340 Warsaw, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0000-5363-7839","authenticated-orcid":false,"given":"Jakub","family":"K\u0119dziorek","sequence":"additional","affiliation":[{"name":"Animallab Veterinary Laboratory, \u015arodkowa 2\/4, 03-340 Warsaw, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0007-0013-3056","authenticated-orcid":false,"given":"Mateusz","family":"Antecki","sequence":"additional","affiliation":[{"name":"Animallab Veterinary Laboratory, \u015arodkowa 2\/4, 03-340 Warsaw, Poland"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,7,11]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"3195","DOI":"10.1128\/jcm.30.12.3195-3199.1992","article-title":"The magnetic immuno polymerase chain reaction assay for direct detection of salmonellae in fecal samples","volume":"30","author":"Widjojoatmodjo","year":"1992","journal-title":"J. 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