A new protocol for absolute quantification of haemosporidian parasites in raptors and comparison with current assays
Abstract Background Accurate quantification of infection intensity is essential to estimate infection patterns of avian haemosporidian parasites in order to understand the evolution of host-parasite associations. Traditional microscopy is cost-effective but requires high-quality blood smears and con...
| Main Authors: | , , , , , , , , |
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| Format: | Article |
| Language: | English |
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BMC
2020-07-01
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| Series: | Parasites & Vectors |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s13071-020-04195-y |
| _version_ | 1819133781714403328 |
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| author | Xi Huang Di Huang Yuge Liang Linlin Zhang Guocheng Yang Boye Liu Yangyang Peng Wenhong Deng Lu Dong |
| author_facet | Xi Huang Di Huang Yuge Liang Linlin Zhang Guocheng Yang Boye Liu Yangyang Peng Wenhong Deng Lu Dong |
| author_sort | Xi Huang |
| collection | DOAJ |
| description | Abstract Background Accurate quantification of infection intensity is essential to estimate infection patterns of avian haemosporidian parasites in order to understand the evolution of host-parasite associations. Traditional microscopy is cost-effective but requires high-quality blood smears and considerable experience, while the widely used semi-quantitative qPCR methods are mostly employed with ideal, laboratory-based golden samples and standard curves, which may limit the comparison of parasitemia from different laboratories. Methods Here we present a digital droplet PCR (ddPCR) protocol for absolute quantification of avian haemosporidians in raptors. Novel primers were designed that target a conserved fragment of a rRNA region of the mitochondrial genome of the parasites. Sensitivity and repeatability were evaluated compared to qPCR and other assays. Results This ddPCR assay enables accurate quantification of haemosporidian parasites belonging to Plasmodium, Haemoproteus and Leucocytozoon with minimum infection quantities of 10−5 (i.e. one parasite copy in 105 host genomes) without the use of standard curves. Quantities assessed by ddPCR were more accurate than qPCR using the same primers through reduction of non-specific amplification in low-intensity samples. The ddPCR technique was more consistent among technical duplicates and reactions, especially when infection intensities were low, and this technique demonstrated equal sensitivity with high correspondence (R 2 = 0.97) compared to the widely used qPCR assay. Both ddPCR and qPCR identified more positive samples than the standard nested PCR protocol for the cytb gene used for barcoding avian haemosporidians. Conclusions We developed a novel ddPCR assay enabling accurate quantification of avian haemosporidians without golden samples or standard curves. This assay can be used as a robust method for investigating infection patterns in different host-parasite assemblages and can facilitate the comparison of results from different laboratories. |
| first_indexed | 2024-12-22T09:52:45Z |
| format | Article |
| id | doaj.art-1cd36ee9bf5542e1962768c10222a897 |
| institution | Directory Open Access Journal |
| issn | 1756-3305 |
| language | English |
| last_indexed | 2024-12-22T09:52:45Z |
| publishDate | 2020-07-01 |
| publisher | BMC |
| record_format | Article |
| series | Parasites & Vectors |
| spelling | doaj.art-1cd36ee9bf5542e1962768c10222a8972022-12-21T18:30:21ZengBMCParasites & Vectors1756-33052020-07-011311910.1186/s13071-020-04195-yA new protocol for absolute quantification of haemosporidian parasites in raptors and comparison with current assaysXi Huang0Di Huang1Yuge Liang2Linlin Zhang3Guocheng Yang4Boye Liu5Yangyang Peng6Wenhong Deng7Lu Dong8College of Life Sciences, Beijing Normal UniversityCollege of Life Sciences, Beijing Normal UniversityCollege of Life Sciences, Beijing Normal UniversityCollege of Life Sciences, Beijing Normal UniversityCollege of Life Sciences, Beijing Normal UniversityShaanxi Institute of ZoologyCollege of Life Sciences, Beijing Normal UniversityCollege of Life Sciences, Beijing Normal UniversityCollege of Life Sciences, Beijing Normal UniversityAbstract Background Accurate quantification of infection intensity is essential to estimate infection patterns of avian haemosporidian parasites in order to understand the evolution of host-parasite associations. Traditional microscopy is cost-effective but requires high-quality blood smears and considerable experience, while the widely used semi-quantitative qPCR methods are mostly employed with ideal, laboratory-based golden samples and standard curves, which may limit the comparison of parasitemia from different laboratories. Methods Here we present a digital droplet PCR (ddPCR) protocol for absolute quantification of avian haemosporidians in raptors. Novel primers were designed that target a conserved fragment of a rRNA region of the mitochondrial genome of the parasites. Sensitivity and repeatability were evaluated compared to qPCR and other assays. Results This ddPCR assay enables accurate quantification of haemosporidian parasites belonging to Plasmodium, Haemoproteus and Leucocytozoon with minimum infection quantities of 10−5 (i.e. one parasite copy in 105 host genomes) without the use of standard curves. Quantities assessed by ddPCR were more accurate than qPCR using the same primers through reduction of non-specific amplification in low-intensity samples. The ddPCR technique was more consistent among technical duplicates and reactions, especially when infection intensities were low, and this technique demonstrated equal sensitivity with high correspondence (R 2 = 0.97) compared to the widely used qPCR assay. Both ddPCR and qPCR identified more positive samples than the standard nested PCR protocol for the cytb gene used for barcoding avian haemosporidians. Conclusions We developed a novel ddPCR assay enabling accurate quantification of avian haemosporidians without golden samples or standard curves. This assay can be used as a robust method for investigating infection patterns in different host-parasite assemblages and can facilitate the comparison of results from different laboratories.http://link.springer.com/article/10.1186/s13071-020-04195-yAvian haemosporidiaddPCRqPCRInfection intensityRaptor |
| spellingShingle | Xi Huang Di Huang Yuge Liang Linlin Zhang Guocheng Yang Boye Liu Yangyang Peng Wenhong Deng Lu Dong A new protocol for absolute quantification of haemosporidian parasites in raptors and comparison with current assays Parasites & Vectors Avian haemosporidia ddPCR qPCR Infection intensity Raptor |
| title | A new protocol for absolute quantification of haemosporidian parasites in raptors and comparison with current assays |
| title_full | A new protocol for absolute quantification of haemosporidian parasites in raptors and comparison with current assays |
| title_fullStr | A new protocol for absolute quantification of haemosporidian parasites in raptors and comparison with current assays |
| title_full_unstemmed | A new protocol for absolute quantification of haemosporidian parasites in raptors and comparison with current assays |
| title_short | A new protocol for absolute quantification of haemosporidian parasites in raptors and comparison with current assays |
| title_sort | new protocol for absolute quantification of haemosporidian parasites in raptors and comparison with current assays |
| topic | Avian haemosporidia ddPCR qPCR Infection intensity Raptor |
| url | http://link.springer.com/article/10.1186/s13071-020-04195-y |
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