A new real-time PCR protocol for detection of avian haemosporidians

Particle and Fibre Toxicology

Vasyl V. Tkach¹  Alan Fecchio²  Jason D. Weckstein²  Jeffrey A. Bell¹ 

[1]Department of Biology, University of North Dakota, 10 Cornell Street STOP 9019, Grand Forks 58202, ND, USA

[2]Department of Ornithology and Department of Biodiversity, Earth, and Environmental Sciences, Academy of Natural Sciences of Drexel University, 1900 Benjamin Franklin Parkway, Philadelphia 19103, PA, USA

关键词: Real-time PCR;    Leucocytozoon;    Haemoproteus;    Plasmodium;    Avian haemosporidians;   

Others  :  1222201
DOI  :  10.1186/s13071-015-0993-0

 received in 2015-05-31, accepted in 2015-07-07,  发布年份 2015

【 摘 要 】

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 (χ²  = 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.

【 授权许可】

   
2015 Bell et al.

【 图 表 】

Fig. 1.

Fig. 2.

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