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A new one-step multiplex PCR assay for simultaneous detection and identification of avian haemosporidian parasites

Ciloglu, Arif LU ; Ellis, Vincenzo A. LU ; Bernotienė, Rasa; Valkiūnas, Gediminas and Bensch, Staffan LU (2019) In Parasitology Research 118(1). p.191-201
Abstract

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... (More)

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.

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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
Haemoproteus, Leucocytozoon, Mixed infection, Multiplex PCR, Parasite detection, Plasmodium
in
Parasitology Research
volume
118
issue
1
pages
191 - 201
publisher
Springer
external identifiers
  • scopus:85058079648
ISSN
0932-0113
DOI
10.1007/s00436-018-6153-7
language
English
LU publication?
yes
id
39747669-946d-48f8-ba27-c653a6a454cf
date added to LUP
2019-01-07 15:58:01
date last changed
2019-02-20 11:41:51
@article{39747669-946d-48f8-ba27-c653a6a454cf,
  abstract     = {<p>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.</p>},
  author       = {Ciloglu, Arif and Ellis, Vincenzo A. and Bernotienė, Rasa and Valkiūnas, Gediminas and Bensch, Staffan},
  issn         = {0932-0113},
  keyword      = {Haemoproteus,Leucocytozoon,Mixed infection,Multiplex PCR,Parasite detection,Plasmodium},
  language     = {eng},
  number       = {1},
  pages        = {191--201},
  publisher    = {Springer},
  series       = {Parasitology Research},
  title        = {A new one-step multiplex PCR assay for simultaneous detection and identification of avian haemosporidian parasites},
  url          = {http://dx.doi.org/10.1007/s00436-018-6153-7},
  volume       = {118},
  year         = {2019},
}