An ultra-simplified protocol for PCR template preparation from both unsporulated and sporulated Eimeria oocysts
Molecular biological techniques have enabled the accurate identification of the avian Eimeria parasite, however, the preparation of PCR template remains a bottleneck due to contaminants from feces and the robust oocyst's wall resistant to chemical and mechanical force. Generally, the preparatio...
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Elsevier
2025-03-01
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| Series: | Poultry Science |
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| Online Access: | http://www.sciencedirect.com/science/article/pii/S0032579125000471 |
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| author | Aruto Takano Dennis V. Umali April H. Wardhana Dyah H. Sawitri Isao Teramoto Toshimitsu Hatabu Yasutoshi Kido Akira Kaneko Kazumi Sasai Hiromitsu Katoh Makoto Matsubayashi |
| author_facet | Aruto Takano Dennis V. Umali April H. Wardhana Dyah H. Sawitri Isao Teramoto Toshimitsu Hatabu Yasutoshi Kido Akira Kaneko Kazumi Sasai Hiromitsu Katoh Makoto Matsubayashi |
| author_sort | Aruto Takano |
| collection | DOAJ |
| description | Molecular biological techniques have enabled the accurate identification of the avian Eimeria parasite, however, the preparation of PCR template remains a bottleneck due to contaminants from feces and the robust oocyst's wall resistant to chemical and mechanical force. Generally, the preparation of PCR template involves three main steps: (1) pretreatment of oocysts; (2) disruption of oocysts; and (3) purification of genomic DNA. We prepared PCR templates from both unsporulated and sporulated E. tenella oocysts using various protocols, followed by species-specific PCR to define the limit of detection. Our data revealed that whereas neither pretreatment of oocysts with sodium hypochlorite nor purification of genomic DNA with commercial kits improved the limit of detection of PCR, disruption of oocysts was a critical step in the preparation of PCR templates. The most sensitive PCR assay was achieved with the template prepared by disrupting oocysts suspended in distilled water, followed by bead-beating and heating at 99°C for 5 min, which detected 0.16 oocysts per PCR. This ultra-simplified protocol for preparation of PCR template, which does not require expensive reagents or equipment, will significantly enhance the sensitive and efficient molecular identification of Eimeria. It will improve our understanding of the prevalence of this parasite at the species level and contribute to the development of techniques for the control in the field. |
| format | Article |
| id | doaj-art-f92b09c12e7d41ed9f4a25d30a7888e5 |
| institution | Kabale University |
| issn | 0032-5791 |
| language | English |
| publishDate | 2025-03-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Poultry Science |
| spelling | doaj-art-f92b09c12e7d41ed9f4a25d30a7888e52025-02-03T04:16:26ZengElsevierPoultry Science0032-57912025-03-011043104810An ultra-simplified protocol for PCR template preparation from both unsporulated and sporulated Eimeria oocystsAruto Takano0Dennis V. Umali1April H. Wardhana2Dyah H. Sawitri3Isao Teramoto4Toshimitsu Hatabu5Yasutoshi Kido6Akira Kaneko7Kazumi Sasai8Hiromitsu Katoh9Makoto Matsubayashi10Departments of Veterinary Immunology, Graduate School of Veterinary Medical Sciences, Osaka Metropolitan University, Osaka, 598-8531, Japan; Tri-Ace Co., Ltd., Setagaya, Tokyo, 157-0066, JapanDepartment of Veterinary Clinical Sciences, College of Veterinary Medicine, University of the Philippines Los Baños, College, Laguna, 4031, PhilippinesResearch Center for Veterinary Science, National Research and Innovation Agency, Bogor, 16911, Indonesia; Indonesia Research Center for Veterinary Science, the Indonesian Agency for Agricultural Research and Development, Ministry of Agriculture, JakartaResearch Center for Veterinary Science, National Research and Innovation Agency, Bogor, 16911, Indonesia; Indonesia Research Center for Veterinary Science, the Indonesian Agency for Agricultural Research and Development, Ministry of Agriculture, JakartaDepartments of Virology and Parasitology, Graduate School of Medicine, Osaka Metropolitan University, Osaka, 545-8585, JapanLaboratory of Animal Physiology, Graduate School of Environmental, Life, Natural Science and Technology, Okayama University, Okayama, 700-8530, JapanDepartments of Virology and Parasitology, Graduate School of Medicine, Osaka Metropolitan University, Osaka, 545-8585, Japan; Osaka International Research Center for Infectious Diseases, Osaka Metropolitan University, Osaka, 545-8585, JapanDepartments of Virology and Parasitology, Graduate School of Medicine, Osaka Metropolitan University, Osaka, 545-8585, Japan; Osaka International Research Center for Infectious Diseases, Osaka Metropolitan University, Osaka, 545-8585, JapanDepartments of Veterinary Immunology, Graduate School of Veterinary Medical Sciences, Osaka Metropolitan University, Osaka, 598-8531, JapanDepartments of Veterinary Immunology, Graduate School of Veterinary Medical Sciences, Osaka Metropolitan University, Osaka, 598-8531, Japan; Tri-Ace Co., Ltd., Setagaya, Tokyo, 157-0066, Japan; Department of Veterinary Clinical Sciences, College of Veterinary Medicine, University of the Philippines Los Baños, College, Laguna, 4031, PhilippinesDepartments of Veterinary Immunology, Graduate School of Veterinary Medical Sciences, Osaka Metropolitan University, Osaka, 598-8531, Japan; Osaka International Research Center for Infectious Diseases, Osaka Metropolitan University, Osaka, 545-8585, Japan; Corresponding author at: Address: Graduate School of Veterinary Sciences, Osaka Metropolitan University, 1-58 Rinku Orai Kita, Izumisano, Osaka, 598-8531, Japan.Molecular biological techniques have enabled the accurate identification of the avian Eimeria parasite, however, the preparation of PCR template remains a bottleneck due to contaminants from feces and the robust oocyst's wall resistant to chemical and mechanical force. Generally, the preparation of PCR template involves three main steps: (1) pretreatment of oocysts; (2) disruption of oocysts; and (3) purification of genomic DNA. We prepared PCR templates from both unsporulated and sporulated E. tenella oocysts using various protocols, followed by species-specific PCR to define the limit of detection. Our data revealed that whereas neither pretreatment of oocysts with sodium hypochlorite nor purification of genomic DNA with commercial kits improved the limit of detection of PCR, disruption of oocysts was a critical step in the preparation of PCR templates. The most sensitive PCR assay was achieved with the template prepared by disrupting oocysts suspended in distilled water, followed by bead-beating and heating at 99°C for 5 min, which detected 0.16 oocysts per PCR. This ultra-simplified protocol for preparation of PCR template, which does not require expensive reagents or equipment, will significantly enhance the sensitive and efficient molecular identification of Eimeria. It will improve our understanding of the prevalence of this parasite at the species level and contribute to the development of techniques for the control in the field.http://www.sciencedirect.com/science/article/pii/S0032579125000471Coccidian parasiteEimeria tenellaExtractionMolecular identificationOocyst |
| spellingShingle | Aruto Takano Dennis V. Umali April H. Wardhana Dyah H. Sawitri Isao Teramoto Toshimitsu Hatabu Yasutoshi Kido Akira Kaneko Kazumi Sasai Hiromitsu Katoh Makoto Matsubayashi An ultra-simplified protocol for PCR template preparation from both unsporulated and sporulated Eimeria oocysts Poultry Science Coccidian parasite Eimeria tenella Extraction Molecular identification Oocyst |
| title | An ultra-simplified protocol for PCR template preparation from both unsporulated and sporulated Eimeria oocysts |
| title_full | An ultra-simplified protocol for PCR template preparation from both unsporulated and sporulated Eimeria oocysts |
| title_fullStr | An ultra-simplified protocol for PCR template preparation from both unsporulated and sporulated Eimeria oocysts |
| title_full_unstemmed | An ultra-simplified protocol for PCR template preparation from both unsporulated and sporulated Eimeria oocysts |
| title_short | An ultra-simplified protocol for PCR template preparation from both unsporulated and sporulated Eimeria oocysts |
| title_sort | ultra simplified protocol for pcr template preparation from both unsporulated and sporulated eimeria oocysts |
| topic | Coccidian parasite Eimeria tenella Extraction Molecular identification Oocyst |
| url | http://www.sciencedirect.com/science/article/pii/S0032579125000471 |
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