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Despite significant advances in vaccination and new therapeutic agents, little attention has been paid to validating methods for the diagnosis of fascioliasis in humans. Serological techniques are convenient assays that significantly improves the diagnosis of <jats:italic>Fasciola<\/jats:italic> infection. However, a more sensitive method is required. The aim of this study was to compare the Real-Time PCR technique with the indirect-ELISA for the detection of <jats:italic>Fasciola hepatica<\/jats:italic> in human. Using a panel of sera from patients infected with <jats:italic>Fasciola hepatica<\/jats:italic> (<jats:italic>n<\/jats:italic>\u2009=\u200951), other parasitic infections (<jats:italic>n<\/jats:italic>\u2009=\u20097), and uninfected controls (<jats:italic>n<\/jats:italic>\u2009=\u200912), we optimized an ELISA which employs an excretory\u2013secretory antigens from <jats:italic>F. hepatica<\/jats:italic> for the detection of human fascioliasis. After DNA extraction from the samples, molecular analysis was done using Real-Time PCR technique based on the <jats:italic>Fasciola<\/jats:italic> ribosomal ITS1 sequence. Of 70 patient serum samples, 44 (62.86%) samples were identified as positive <jats:italic>F. hepatica<\/jats:italic> infection using ELISA and Real-Time PCR assays. There was no cross-reaction with other parasitic diseases such as toxoplasmosis, leishmaniasis, taeniasis, hydatidosis, trichinosis, toxocariasis, and strongyloidiasis. The significant difference between the agreement and similarity of the results of patients with indirect ELISA and Real-Time PCR was 94.4% and 99.2%, respectively (Cohen\u2019s kappa\u2009\u2265\u20090.7; <jats:italic>P<\/jats:italic>\u2009=\u20090.02). Based on the Kappa agreement findings, the significant agreement between the results of ELISA and Real-Time PCR indicates the accuracy and reliability of these tests in the diagnosis of <jats:italic>F. hepatica<\/jats:italic> in humans.<\/jats:p>","DOI":"10.1038\/s41598-024-54602-y","type":"journal-article","created":{"date-parts":[[2024,2,16]],"date-time":"2024-02-16T06:01:59Z","timestamp":1708063319000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":7,"title":["Comparative evaluation of real-time PCR and ELISA for the detection of human fascioliasis"],"prefix":"10.1038","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0009-0000-2197-1285","authenticated-orcid":false,"given":"Fatemeh","family":"Bakhshipour","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2265-7460","authenticated-orcid":false,"given":"Mohammad","family":"Zibaei","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1048-2512","authenticated-orcid":false,"given":"Mohammad Bagher","family":"Rokni","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8359-7452","authenticated-orcid":false,"given":"Abolfazl","family":"Miahipour","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4026-4110","authenticated-orcid":false,"given":"Farzaneh","family":"Firoozeh","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3988-239X","authenticated-orcid":false,"given":"Masoud","family":"Beheshti","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Leila","family":"Beikzadeh","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3664-4548","authenticated-orcid":false,"given":"Gita","family":"Alizadeh","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mojgan","family":"Aryaeipour","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0504-2982","authenticated-orcid":false,"given":"Vahid","family":"Raissi","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2024,2,16]]},"reference":[{"key":"54602_CR1","doi-asserted-by":"publisher","first-page":"403","DOI":"10.3347\/kjp.1991.29.4.403","volume":"29","author":"EC Chang","year":"1991","unstructured":"Chang, E. 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