A new real-time PCR protocol for detection of avian haemosporidians
Abstract Background Birds possess the most diverse assemblage of haemosporidian parasites; including three genera, Plasmodium, Haemoproteus, and Leucocytozoon. Currently there are over 200 morphologically identified avian haemosporidian species, although true species richness is unknown due to great...
| Main Authors: | , , , |
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| Format: | Article |
| Language: | English |
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BMC
2015-07-01
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| Series: | Parasites & Vectors |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s13071-015-0993-0 |
| _version_ | 1827933798221742080 |
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| author | Jeffrey A. Bell Jason D. Weckstein Alan Fecchio Vasyl V. Tkach |
| author_facet | Jeffrey A. Bell Jason D. Weckstein Alan Fecchio Vasyl V. Tkach |
| author_sort | Jeffrey A. Bell |
| collection | DOAJ |
| description | Abstract Background Birds possess the most diverse assemblage of haemosporidian parasites; including three genera, Plasmodium, Haemoproteus, and Leucocytozoon. Currently there are over 200 morphologically identified avian haemosporidian species, although true species richness is unknown due to great genetic diversity and insufficient sampling in highly diverse regions. Studies aimed at surveying haemosporidian diversity involve collecting and screening samples from hundreds to thousands of individuals. Currently, screening relies on microscopy and/or single or nested standard PCR. Although effective, these methods are time and resource consuming, and in the case of microscopy require substantial expertise. Here we report a newly developed real-time PCR protocol designed to quickly and reliably detect all three genera of avian haemosporidians in a single biochemical reaction. Methods Using available DNA sequences from avian haemosporidians we designed primers R330F and R480RL, which flank a 182 base pair fragment of mitochondrial conserved rDNA. These primers were initially tested using real-time PCR on samples from Malawi, Africa, previously screened for avian haemosporidians using traditional nested PCR. Our real time protocol was further tested on 94 samples from the Cerrado biome of Brazil, previously screened using a single PCR assay for haemosporidian parasites. These samples were also amplified using modified nested PCR protocols, allowing for comparisons between the three different screening methods (single PCR, nested PCR, real-time PCR). Results The real-time PCR protocol successfully identified all three genera of avian haemosporidians from both single and mixed infections previously detected from Malawi. There was no significant difference between the three different screening protocols used for the 94 samples from the Brazilian Cerrado (χ 2 = 0.3429, df = 2, P = 0.842). After proving effective, the real-time protocol was used to screen 2113 Brazilian samples, identifying 693 positive samples. Conclusions Our real-time PCR assay proved as effective as two widely used molecular screening techniques, single PCR and nested PCR. However, the real-time protocol has the distinct advantage of detecting all three genera in a single reaction, which significantly increases efficiency by greatly decreasing screening time and cost. Our real-time PCR protocol is therefore a valuable tool in the quickly expanding field of avian haemosporidian research. |
| first_indexed | 2024-03-13T07:29:50Z |
| format | Article |
| id | doaj.art-cb92c05e9cca41cb954bec36c36b2bad |
| institution | Directory Open Access Journal |
| issn | 1756-3305 |
| language | English |
| last_indexed | 2024-03-13T07:29:50Z |
| publishDate | 2015-07-01 |
| publisher | BMC |
| record_format | Article |
| series | Parasites & Vectors |
| spelling | doaj.art-cb92c05e9cca41cb954bec36c36b2bad2023-06-04T11:10:20ZengBMCParasites & Vectors1756-33052015-07-01811910.1186/s13071-015-0993-0A new real-time PCR protocol for detection of avian haemosporidiansJeffrey A. Bell0Jason D. Weckstein1Alan Fecchio2Vasyl V. Tkach3Department of Biology, University of North DakotaDepartment of Ornithology and Department of Biodiversity, Earth, and Environmental Sciences, Academy of Natural Sciences of Drexel UniversityDepartment of Ornithology and Department of Biodiversity, Earth, and Environmental Sciences, Academy of Natural Sciences of Drexel UniversityDepartment of Biology, University of North DakotaAbstract Background Birds possess the most diverse assemblage of haemosporidian parasites; including three genera, Plasmodium, Haemoproteus, and Leucocytozoon. Currently there are over 200 morphologically identified avian haemosporidian species, although true species richness is unknown due to great genetic diversity and insufficient sampling in highly diverse regions. Studies aimed at surveying haemosporidian diversity involve collecting and screening samples from hundreds to thousands of individuals. Currently, screening relies on microscopy and/or single or nested standard PCR. Although effective, these methods are time and resource consuming, and in the case of microscopy require substantial expertise. Here we report a newly developed real-time PCR protocol designed to quickly and reliably detect all three genera of avian haemosporidians in a single biochemical reaction. Methods Using available DNA sequences from avian haemosporidians we designed primers R330F and R480RL, which flank a 182 base pair fragment of mitochondrial conserved rDNA. These primers were initially tested using real-time PCR on samples from Malawi, Africa, previously screened for avian haemosporidians using traditional nested PCR. Our real time protocol was further tested on 94 samples from the Cerrado biome of Brazil, previously screened using a single PCR assay for haemosporidian parasites. These samples were also amplified using modified nested PCR protocols, allowing for comparisons between the three different screening methods (single PCR, nested PCR, real-time PCR). Results The real-time PCR protocol successfully identified all three genera of avian haemosporidians from both single and mixed infections previously detected from Malawi. There was no significant difference between the three different screening protocols used for the 94 samples from the Brazilian Cerrado (χ 2 = 0.3429, df = 2, P = 0.842). After proving effective, the real-time protocol was used to screen 2113 Brazilian samples, identifying 693 positive samples. Conclusions Our real-time PCR assay proved as effective as two widely used molecular screening techniques, single PCR and nested PCR. However, the real-time protocol has the distinct advantage of detecting all three genera in a single reaction, which significantly increases efficiency by greatly decreasing screening time and cost. Our real-time PCR protocol is therefore a valuable tool in the quickly expanding field of avian haemosporidian research.https://doi.org/10.1186/s13071-015-0993-0Avian haemosporidiansPlasmodiumHaemoproteusLeucocytozoonReal-time PCR |
| spellingShingle | Jeffrey A. Bell Jason D. Weckstein Alan Fecchio Vasyl V. Tkach A new real-time PCR protocol for detection of avian haemosporidians Parasites & Vectors Avian haemosporidians Plasmodium Haemoproteus Leucocytozoon Real-time PCR |
| title | A new real-time PCR protocol for detection of avian haemosporidians |
| title_full | A new real-time PCR protocol for detection of avian haemosporidians |
| title_fullStr | A new real-time PCR protocol for detection of avian haemosporidians |
| title_full_unstemmed | A new real-time PCR protocol for detection of avian haemosporidians |
| title_short | A new real-time PCR protocol for detection of avian haemosporidians |
| title_sort | new real time pcr protocol for detection of avian haemosporidians |
| topic | Avian haemosporidians Plasmodium Haemoproteus Leucocytozoon Real-time PCR |
| url | https://doi.org/10.1186/s13071-015-0993-0 |
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