{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,1]],"date-time":"2026-08-01T01:44:07Z","timestamp":1785548647124,"version":"3.56.0"},"reference-count":35,"publisher":"MDPI AG","issue":"1","license":[{"start":{"date-parts":[[2022,12,21]],"date-time":"2022-12-21T00:00:00Z","timestamp":1671580800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100001659","name":"German Research Foundation (DFG) through the project \u201cTungiasis in East-Africa","doi-asserted-by":"publisher","award":["405027164; KR 2245\/7-1"],"award-info":[{"award-number":["405027164; KR 2245\/7-1"]}],"id":[{"id":"10.13039\/501100001659","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Swedish International Development Cooperation Agency (Sida)","award":["405027164; KR 2245\/7-1"],"award-info":[{"award-number":["405027164; KR 2245\/7-1"]}]},{"name":"the Swedish International Development Cooperation Agency (Sida)","award":["405027164; KR 2245\/7-1"],"award-info":[{"award-number":["405027164; KR 2245\/7-1"]}]},{"name":"Swiss Agency for Development and Cooperation (SDC)","award":["405027164; KR 2245\/7-1"],"award-info":[{"award-number":["405027164; KR 2245\/7-1"]}]},{"name":"Federal Democratic Republic of Ethiopia","award":["405027164; KR 2245\/7-1"],"award-info":[{"award-number":["405027164; KR 2245\/7-1"]}]},{"name":"the Federal Democratic Republic of Ethiopia","award":["405027164; KR 2245\/7-1"],"award-info":[{"award-number":["405027164; KR 2245\/7-1"]}]},{"name":"Government of the Republic of Kenya","award":["405027164; KR 2245\/7-1"],"award-info":[{"award-number":["405027164; KR 2245\/7-1"]}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Insects"],"abstract":"<jats:p>Tungiasis is a neglected tropical disease caused by skin-penetrating female Tunga penetrans fleas. Although tungiasis causes severe health problems, its ecology is poorly understood and morphological descriptions of the larvae are unavailable. To identify T. penetrans immature stages and sites where they develop, diagnostic PCRs are required. However, flea larvae feed on soil organic matter rich in PCR inhibitors. Here, three DNA preparation methods, including a soil DNA kit that removes inhibitors, a simple ammonium acetate precipitation approach (AmAcet) and a crude lysate of larvae (CL), were combined with amplification by the highly processive FIREPol\u00ae Taq or the inhibitor-resistant Phusion\u00ae polymerase. Independent of the polymerase used, the frequency of successful amplification, Cq values and PCR efficacies for the low-cost CL and AmAcet methods were superior to the commercial kit for amplification of a 278 bp partial internal transcribed spacer-2 (ITS-2) and a 730 bp pan-Siphonaptera cytochrome oxidase II PCR. For the CL method combined with Phusion\u00ae polymerase, the costs were approximately 20-fold lower than for the methods based on the soil DNA kit, which is a considerable advantage in resource-poor settings. The ITS-2 PCR did not amplify Ctenocephalides felis genomic or Tunga trimammilata ITS-2 plasmid DNA, meaning it can be used to specifically identify T. penetrans.<\/jats:p>","DOI":"10.3390\/insects14010005","type":"journal-article","created":{"date-parts":[[2022,12,22]],"date-time":"2022-12-22T02:31:11Z","timestamp":1671676271000},"page":"5","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":6,"title":["Cost-Effective PCR-Based Identification of Tunga penetrans (Siphonaptera) Larvae Extracted from Soil Samples Containing PCR Inhibitor-Rich Material"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-5975-0776","authenticated-orcid":false,"given":"Billy L.","family":"Amugune","sequence":"first","affiliation":[{"name":"International Centre of Insect Physiology and Ecology, Human Health Theme, Nairobi 00100, Kenya"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0045-5588","authenticated-orcid":false,"given":"Abneel K.","family":"Matharu","sequence":"additional","affiliation":[{"name":"International Centre of Insect Physiology and Ecology, Human Health Theme, Nairobi 00100, Kenya"},{"name":"Institute for Parasitology and Tropical Veterinary Medicine, Freie Universit\u00e4t Berlin, 14163 Berlin, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Paul","family":"Ouma","sequence":"additional","affiliation":[{"name":"International Centre of Insect Physiology and Ecology, Human Health Theme, Nairobi 00100, Kenya"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Francis","family":"Mutebi","sequence":"additional","affiliation":[{"name":"School of Veterinary Medicine and Animal Resources, College of Veterinary Medicine, Animal Resources and Biosecurity, Makerere University, Kampala 10218, Uganda"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2264-4459","authenticated-orcid":false,"given":"Lynne","family":"Elson","sequence":"additional","affiliation":[{"name":"KEMRI-Wellcome Trust Research Programme, Kilifi 80108, Kenya"},{"name":"Centre for Tropical Medicine and Global Health, Nuffield Department of Medicine, University of Oxford, Oxford OX3 7BN, UK"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4037-431X","authenticated-orcid":false,"given":"Ulrike","family":"Fillinger","sequence":"additional","affiliation":[{"name":"International Centre of Insect Physiology and Ecology, Human Health Theme, Nairobi 00100, Kenya"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2842-8100","authenticated-orcid":false,"given":"J\u00fcrgen","family":"Kr\u00fccken","sequence":"additional","affiliation":[{"name":"Institute for Parasitology and Tropical Veterinary Medicine, Freie Universit\u00e4t Berlin, 14163 Berlin, Germany"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2022,12,21]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"357","DOI":"10.1016\/j.tmaid.2013.10.001","article-title":"Tungiasis\u2014A Janus-Faced Parasitic Skin Disease","volume":"11","author":"Feldmeier","year":"2013","journal-title":"Travel Med. 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