A new one-step multiplex PCR assay for simultaneous detection and identification of avian haemosporidian parasites
Accurate detection and identification are essential components for epidemiological, ecological, and evolutionary surveys of avian haemosporidian parasites. Microscopy has been used for more than 100 years to detect and identify these parasites; however, this technique requires considerable training and high-level expertise. Several PCR methods with highly sensitive and specific detection capabilities have now been developed in addition to microscopic examination. However, recent studies have shown that these molecular protocols are insufficient at detecting mixed infections of different haemosporidian parasite species and genetic lineages. In this study, we developed a simple, sensitive, and specific multiplex PCR assay for simultaneous detection and discrimination of parasites of the genera Plasmodium, Haemoproteus, and Leucocytozoon in single and mixed infections. Relative quantification of parasite DNA using qPCR showed that the multiplex PCR can amplify parasite DNA ranging in concentration over several orders of magnitude. The detection specificity and sensitivity of this new multiplex PCR assay were also tested in two different laboratories using previously screened natural single and mixed infections. These findings show that the multiplex PCR designed here is highly effective at identifying both single and mixed infections from all three genera of avian haemosporidian parasites. We predict that this one-step multiplex PCR assay, being convenient and inexpensive, will become a widely used method for molecular screening of avian haemosporidian parasites.
KeywordsPlasmodium Haemoproteus Leucocytozoon Parasite detection Mixed infection Multiplex PCR
We thank Xi Huang for assisting with quantitative PCR analyses in the laboratory and Tatjana A. Iezhova for microscopic identification of parasites.
This work was supported by the Swedish Research Council (grant 621-2013-4839 to SB) and also partly funded by the Research Council of Lithuania (grant S-MIP-17-27 to RB). VAE was supported by a postdoctoral fellowship from the Carl Tryggers Foundation.
Compliance with ethical standards
Permission for taking blood samples from birds in Sweden was approved by the Malmö/Lund Committee for Animal Experiment Ethics (M45-14). Procedures with birds in Lithuania were performed by licensed researchers and were approved by the Ethical Commission of the Baltic Laboratory Animal Science Association, Lithuania; Lithuanian State Food and Veterinary Office and Environmental Protection Agency, Vilnius.
Conflict of interest
The authors declare that they have no conflict of interest.
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