{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,30]],"date-time":"2026-09-30T03:36:55Z","timestamp":1790739415943,"version":"4.1.0"},"reference-count":49,"publisher":"Wiley","issue":"10","license":[{"start":{"date-parts":[[2025,5,13]],"date-time":"2025-05-13T00:00:00Z","timestamp":1747094400000},"content-version":"vor","delay-in-days":0,"URL":"http:\/\/onlinelibrary.wiley.com\/termsAndConditions#vor"}],"content-domain":{"domain":["onlinelibrary.wiley.com"],"crossmark-restriction":true},"short-container-title":["Eur J Clin Investigation"],"published-print":{"date-parts":[[2025,10]]},"abstract":"<jats:title>Abstract<\/jats:title><jats:sec><jats:title>Background<\/jats:title><jats:p>Loop\u2010mediated isothermal amplification (LAMP) is a nucleic acid amplification method that gained prominence during the early months of the COVID\u201019 pandemic due to its simplicity, sensitivity and robustness. However, this technique is susceptible to non\u2010specific amplifications, raising concerns about false\u2010positive results and reduced diagnostic accuracy. A primary contributor to false\u2010positive testing is primer dimerization, which can theoretically be mitigated by performing reactions at higher temperatures. Unfortunately, the strand\u2010displacing DNA polymerases typically used in LAMP, such as Bst, exhibit reduced efficiency at elevated temperatures. To address this limitation, we hypothesised that naturally occurring thermophilic analogs of Bst may be capable of supporting LAMP at higher temperatures, thereby improving reaction specificity.<\/jats:p><\/jats:sec><jats:sec><jats:title>Methods<\/jats:title><jats:p>Bioinformatics and recombinant enzyme production allowed the identification and synthesis of several Bst analogs. These were tested in real\u2010time LAMP assays to detect diverse targets, in a wide range of reaction temperatures (63\u00b0C\u201375\u00b0C) and in the presence of typical qPCR inhibitors.<\/jats:p><\/jats:sec><jats:sec><jats:title>Results<\/jats:title><jats:p>Three polymerases\u2014Bst_7, Bst_8 and Bst_15\u2014demonstrated exceptional activity and robust stability at higher temperature conditions (up to 72.5\u00b0C), while displaying considerable resistance to common qPCR inhibitors.<\/jats:p><\/jats:sec><jats:sec><jats:title>Conclusions<\/jats:title><jats:p>The identified thermophilic Bst analogs represent a potential solution for the mitigation of non\u2010specific amplification in LAMP, further boosting the application of this technique in molecular diagnostic settings.<\/jats:p><\/jats:sec>","DOI":"10.1111\/eci.70071","type":"journal-article","created":{"date-parts":[[2025,5,13]],"date-time":"2025-05-13T04:14:44Z","timestamp":1747109684000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":8,"title":["Bringing the heat: Thermostable analogs of Bst polymerase allow high\u2010temperature LAMP"],"prefix":"10.1111","volume":"55","author":[{"ORCID":"https:\/\/orcid.org\/0009-0003-4240-066X","authenticated-orcid":false,"given":"Rita Silva Ramos Madureira","family":"Sim\u00f5es","sequence":"first","affiliation":[{"name":"NZYtech \u2013 Genes &amp; Enzymes, Campus do Lumiar  Lisbon Portugal"},{"name":"CIISA \u2013 Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine University of Lisbon  Lisbon Portugal"},{"name":"Associate Laboratory for Animal and Veterinary Sciences (AL4AnimalS)  Lisbon Portugal"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jo\u00e3o Paulo Soeiro Terra","family":"Teodoro","sequence":"additional","affiliation":[{"name":"NZYtech \u2013 Genes &amp; Enzymes, Campus do Lumiar  Lisbon Portugal"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pedro Miguel Bule","family":"Gomes","sequence":"additional","affiliation":[{"name":"CIISA \u2013 Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine University of Lisbon  Lisbon Portugal"},{"name":"Associate Laboratory for Animal and Veterinary Sciences (AL4AnimalS)  Lisbon Portugal"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carlos Mendes Godinho","family":"de Andrade Fontes","sequence":"additional","affiliation":[{"name":"NZYtech \u2013 Genes &amp; Enzymes, Campus do Lumiar  Lisbon Portugal"},{"name":"CIISA \u2013 Centre for Interdisciplinary Research in Animal Health, Faculty of Veterinary Medicine University of Lisbon  Lisbon Portugal"},{"name":"Associate Laboratory for Animal and Veterinary Sciences (AL4AnimalS)  Lisbon Portugal"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"311","published-online":{"date-parts":[[2025,5,13]]},"reference":[{"key":"e_1_2_10_2_1","doi-asserted-by":"publisher","DOI":"10.1261\/rna.076232.120"},{"key":"e_1_2_10_3_1","doi-asserted-by":"publisher","DOI":"10.1016\/j.jpha.2020.02.010"},{"key":"e_1_2_10_4_1","doi-asserted-by":"publisher","DOI":"10.1093\/nar\/28.12.e63"},{"key":"e_1_2_10_5_1","doi-asserted-by":"publisher","DOI":"10.1007\/s12275-015-4656-9"},{"key":"e_1_2_10_6_1","doi-asserted-by":"publisher","DOI":"10.1006\/mcpr.2002.0415"},{"key":"e_1_2_10_7_1","doi-asserted-by":"publisher","DOI":"10.1002\/rmv.593"},{"key":"e_1_2_10_8_1","doi-asserted-by":"publisher","DOI":"10.1134\/S106816202206022X"},{"key":"e_1_2_10_9_1","doi-asserted-by":"publisher","DOI":"10.1096\/fba.2019-00088"},{"key":"e_1_2_10_10_1","doi-asserted-by":"publisher","DOI":"10.3390\/molecules20046048"},{"key":"e_1_2_10_11_1","doi-asserted-by":"publisher","DOI":"10.1016\/S1050-3862(96)80005-2"},{"key":"e_1_2_10_12_1","doi-asserted-by":"publisher","DOI":"10.1101\/gr.2.4.275"},{"key":"e_1_2_10_13_1","doi-asserted-by":"crossref","unstructured":"SteneshJ RoeBA.DNA polymerase from mesophilic and thermophilic bacteria: I. 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