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However, the limited sensitivity and specificity of these techniques have prompted the exploration of various molecular approaches, such as conventional polymerase chain reaction (PCR) and real-time PCR, to detect <jats:italic>O.\u00a0viverrini<\/jats:italic> infection. Recently, a novel technique known as recombinase polymerase amplification (RPA)\u2013clustered regularly interspaced short palindromic repeats (CRISPR)\/CRISPR-associated protein (Cas) (RPA\u2013CRISPR\/Cas) assay was developed as a point-of-care tool for the detection of various pathogens, including viruses and bacteria such as severe acute respiratory syndrome coronavirus 2 and <jats:italic>Mycobacterium tuberculosis<\/jats:italic>. This technology has demonstrated high sensitivity and specificity. Therefore, we developed and used the RPA\u2013CRISPR\/Cas assay to detect <jats:italic>O.\u00a0viverrini<\/jats:italic> infection in field-collected human feces.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Methods<\/jats:title>\n                <jats:p>To detect <jats:italic>O.\u00a0viverrini<\/jats:italic> infection in fecal samples, we developed a CRISPR\/Cas12a (RNA-guided endonuclease) system combined with RPA (<jats:italic>Ov<\/jats:italic>-RPA\u2013CRISPR\/Cas12a). Several fecal samples, both helminth-positive and helminth-negative, were used for the development and optimization of amplification conditions, CRISPR\/Cas detection conditions, detection limits, and specificity of the RPA\u2013CRISPR\/Cas12a assay for detecting <jats:italic>O.\u00a0viverrini<\/jats:italic> infection. The detection results were determined using a real-time PCR system based on fluorescence values. Additionally, as the reporter was labeled with fluorescein, the detection results were visually inspected using an ultraviolet (UV) transilluminator. A receiver operating characteristic curve (ROC) was used to determine the optimal cutoff value for fluorescence detection. The diagnostic performance, including sensitivity and specificity, of the <jats:italic>Ov<\/jats:italic>-RPA\u2013CRISPR\/Cas12a assay was evaluated on the basis of comparison with standard methods.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Results<\/jats:title>\n                <jats:p>The <jats:italic>Ov<\/jats:italic>-RPA\u2013CRISPR\/Cas12a assay exhibited high specificity for detecting <jats:italic>O.\u00a0viverrini<\/jats:italic> DNA. On the basis of the detection limit, the assay could detect <jats:italic>O.\u00a0viverrini<\/jats:italic> DNA at concentrations as low as 10<jats:sup>\u22121<\/jats:sup>\u00a0ng using the real-time PCR system. However, in this method, visual inspection under UV light required a minimum concentration of 1\u00a0ng. To validate the <jats:italic>Ov<\/jats:italic>-RPA\u2013CRISPR\/Cas12a assay, 121 field-collected fecal samples were analyzed. Microscopic examination revealed that 29 samples were positive for <jats:italic>O.\u00a0viverrini<\/jats:italic>-like eggs. Of these, 18 were confirmed as true positives on the basis of the <jats:italic>Ov<\/jats:italic>-RPA\u2013CRISPR\/Cas12a assay and microscopic examination, whereas 11 samples were determined as positive solely via microscopic examination, indicating the possibility of other minute intestinal fluke infections.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Conclusions<\/jats:title>\n                <jats:p>The <jats:italic>Ov<\/jats:italic>-RPA\u2013CRISPR\/Cas12a assay developed in this study can successfully detect <jats:italic>O.\u00a0viverrini<\/jats:italic> infection in field-collected feces. Due to the high specificity of the assay reported in this study, it can be used as an alternative approach to confirm <jats:italic>O.\u00a0viverrini<\/jats:italic> infection, marking an initial step in the development of point-of-care diagnosis.<\/jats:p>\n              <\/jats:sec><jats:sec>\n                <jats:title>Graphical abstract<\/jats:title>\n                \n              <\/jats:sec>","DOI":"10.1186\/s13071-024-06134-7","type":"journal-article","created":{"date-parts":[[2024,2,21]],"date-time":"2024-02-21T13:02:14Z","timestamp":1708520534000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":14,"title":["Ov-RPA\u2013CRISPR\/Cas12a assay for the detection of Opisthorchis viverrini infection in field-collected human feces"],"prefix":"10.1186","volume":"17","author":[{"given":"Orawan","family":"Phuphisut","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Akkarin","family":"Poodeepiyasawat","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Tippayarat","family":"Yoonuan","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Dorn","family":"Watthanakulpanich","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Charin","family":"Thawornkuno","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Onrapak","family":"Reamtong","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Megumi","family":"Sato","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Poom","family":"Adisakwattana","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2024,2,21]]},"reference":[{"key":"6134_CR1","first-page":"341","volume":"100","author":"International Agency for Research on Cancer","year":"2012","unstructured":"International Agency for Research on Cancer. 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