Comparison of a multiplex PCR with DNA barcoding for identification of container breeding mosquito species
Abstract Background Identification of mosquitoes greatly relies on morphological specification. Since some species cannot be distinguished reliably by morphological methods, it is important to incorporate molecular techniques into the diagnostic pipeline. DNA barcoding using Sanger sequencing is cur...
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| Format: | Article |
| Language: | English |
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BMC
2024-04-01
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| Series: | Parasites & Vectors |
| Subjects: | |
| Online Access: | https://doi.org/10.1186/s13071-024-06255-z |
| _version_ | 1797220008231698432 |
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| author | Julia Reichl Christina Prossegger Sarah Petutschnig Maria Sophia Unterköfler Karin Bakran-Lebl Mateusz Markowicz Alexander Indra Hans-Peter Fuehrer |
| author_facet | Julia Reichl Christina Prossegger Sarah Petutschnig Maria Sophia Unterköfler Karin Bakran-Lebl Mateusz Markowicz Alexander Indra Hans-Peter Fuehrer |
| author_sort | Julia Reichl |
| collection | DOAJ |
| description | Abstract Background Identification of mosquitoes greatly relies on morphological specification. Since some species cannot be distinguished reliably by morphological methods, it is important to incorporate molecular techniques into the diagnostic pipeline. DNA barcoding using Sanger sequencing is currently widely used for identification of mosquito species. However, this method does not allow detection of multiple species in one sample, which would be important when analysing mosquito eggs. Detection of container breeding Aedes is typically performed by collecting eggs using ovitraps. These traps consist of a black container filled with water and a wooden spatula inserted for oviposition support. Aedes mosquitoes of different species might lay single or multiple eggs on the spatula. In contrast to Sanger sequencing of specific polymerase chain reaction (PCR) products, multiplex PCR protocols targeting specific species of interest can be of advantage for detection of multiple species in the same sample. Methods For this purpose, we adapted a previously published PCR protocol for simultaneous detection of four different Aedes species that are relevant for Austrian monitoring programmes, as they can be found in ovitraps: Aedes albopictus, Aedes japonicus, Aedes koreicus, and Aedes geniculatus. For evaluation of the multiplex PCR protocol, we analysed 2271 ovitrap mosquito samples from the years 2021 and 2022, which were collected within the scope of an Austrian nationwide monitoring programme. We compared the results of the multiplex PCR to the results of DNA barcoding. Results Of 2271 samples, the multiplex PCR could identify 1990 samples, while species determination using DNA barcoding of the mitochondrial cytochrome c oxidase subunit I gene was possible in 1722 samples. The multiplex PCR showed a mixture of different species in 47 samples, which could not be detected with DNA barcoding. Conclusions In conclusion, identification of Aedes species in ovitrap samples was more successful when using the multiplex PCR protocol as opposed to the DNA barcoding protocol. Additionally, the multiplex PCR allowed us to detect multiple species in the same sample, while those species might have been missed when using DNA barcoding with Sanger sequencing alone. Therefore, we propose that the multiplex PCR protocol is highly suitable and of great advantage when analysing mosquito eggs from ovitraps. Graphical Abstract |
| first_indexed | 2024-04-24T12:42:42Z |
| format | Article |
| id | doaj.art-8b0fb3fd2a6a4362b3f286fdc3eb8aac |
| institution | Directory Open Access Journal |
| issn | 1756-3305 |
| language | English |
| last_indexed | 2024-04-24T12:42:42Z |
| publishDate | 2024-04-01 |
| publisher | BMC |
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| series | Parasites & Vectors |
| spelling | doaj.art-8b0fb3fd2a6a4362b3f286fdc3eb8aac2024-04-07T11:10:09ZengBMCParasites & Vectors1756-33052024-04-011711910.1186/s13071-024-06255-zComparison of a multiplex PCR with DNA barcoding for identification of container breeding mosquito speciesJulia Reichl0Christina Prossegger1Sarah Petutschnig2Maria Sophia Unterköfler3Karin Bakran-Lebl4Mateusz Markowicz5Alexander Indra6Hans-Peter Fuehrer7Institute for Medical Microbiology and Hygiene, Austrian Agency for Health and Food Safety (AGES)Institute of Parasitology, Department of Pathobiology, University of Veterinary Medicine ViennaInstitute for Medical Microbiology and Hygiene, Austrian Agency for Health and Food Safety (AGES)Institute of Parasitology, Department of Pathobiology, University of Veterinary Medicine ViennaInstitute for Medical Microbiology and Hygiene, Austrian Agency for Health and Food Safety (AGES)Institute for Medical Microbiology and Hygiene, Austrian Agency for Health and Food Safety (AGES)Institute for Medical Microbiology and Hygiene, Austrian Agency for Health and Food Safety (AGES)Institute of Parasitology, Department of Pathobiology, University of Veterinary Medicine ViennaAbstract Background Identification of mosquitoes greatly relies on morphological specification. Since some species cannot be distinguished reliably by morphological methods, it is important to incorporate molecular techniques into the diagnostic pipeline. DNA barcoding using Sanger sequencing is currently widely used for identification of mosquito species. However, this method does not allow detection of multiple species in one sample, which would be important when analysing mosquito eggs. Detection of container breeding Aedes is typically performed by collecting eggs using ovitraps. These traps consist of a black container filled with water and a wooden spatula inserted for oviposition support. Aedes mosquitoes of different species might lay single or multiple eggs on the spatula. In contrast to Sanger sequencing of specific polymerase chain reaction (PCR) products, multiplex PCR protocols targeting specific species of interest can be of advantage for detection of multiple species in the same sample. Methods For this purpose, we adapted a previously published PCR protocol for simultaneous detection of four different Aedes species that are relevant for Austrian monitoring programmes, as they can be found in ovitraps: Aedes albopictus, Aedes japonicus, Aedes koreicus, and Aedes geniculatus. For evaluation of the multiplex PCR protocol, we analysed 2271 ovitrap mosquito samples from the years 2021 and 2022, which were collected within the scope of an Austrian nationwide monitoring programme. We compared the results of the multiplex PCR to the results of DNA barcoding. Results Of 2271 samples, the multiplex PCR could identify 1990 samples, while species determination using DNA barcoding of the mitochondrial cytochrome c oxidase subunit I gene was possible in 1722 samples. The multiplex PCR showed a mixture of different species in 47 samples, which could not be detected with DNA barcoding. Conclusions In conclusion, identification of Aedes species in ovitrap samples was more successful when using the multiplex PCR protocol as opposed to the DNA barcoding protocol. Additionally, the multiplex PCR allowed us to detect multiple species in the same sample, while those species might have been missed when using DNA barcoding with Sanger sequencing alone. Therefore, we propose that the multiplex PCR protocol is highly suitable and of great advantage when analysing mosquito eggs from ovitraps. Graphical Abstracthttps://doi.org/10.1186/s13071-024-06255-zMultiplex PCRAedesAedes albopictusMosquitoesAustriaMonitoring |
| spellingShingle | Julia Reichl Christina Prossegger Sarah Petutschnig Maria Sophia Unterköfler Karin Bakran-Lebl Mateusz Markowicz Alexander Indra Hans-Peter Fuehrer Comparison of a multiplex PCR with DNA barcoding for identification of container breeding mosquito species Parasites & Vectors Multiplex PCR Aedes Aedes albopictus Mosquitoes Austria Monitoring |
| title | Comparison of a multiplex PCR with DNA barcoding for identification of container breeding mosquito species |
| title_full | Comparison of a multiplex PCR with DNA barcoding for identification of container breeding mosquito species |
| title_fullStr | Comparison of a multiplex PCR with DNA barcoding for identification of container breeding mosquito species |
| title_full_unstemmed | Comparison of a multiplex PCR with DNA barcoding for identification of container breeding mosquito species |
| title_short | Comparison of a multiplex PCR with DNA barcoding for identification of container breeding mosquito species |
| title_sort | comparison of a multiplex pcr with dna barcoding for identification of container breeding mosquito species |
| topic | Multiplex PCR Aedes Aedes albopictus Mosquitoes Austria Monitoring |
| url | https://doi.org/10.1186/s13071-024-06255-z |
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