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A cautionary note on the use of nested-PCR for parasite screening - an example from avian blood parasites

Söllözi, Eszter; Hellgren, Olof LU and Hasselquist, Dennis LU (2008) In Journal of Parasitology 94(2). p.562-564
Abstract
The use of new powerful nested polymerase chain reaction (PCR) techniques to identify and screen for prevalence of parasites has a huge potential. It allows for the detection and identification of low-intensity infections, but its high sensitivity and technical setup may also induce problems. Here, we report a cautionary note regarding misleading amplification of avian malaria species (Haemoproteus and Plasmodium) during Leucocytozoon spp. detection. We used a previously described nested PCR method for the molecular detection of avian malaria and Leucocytozoon spp. In the first step of the PCR protocol, these parasites are detected simultaneously; in the second PCR, Haemoproteus and Plasmodium spp. are separated from Leucocytozoon spp.... (More)
The use of new powerful nested polymerase chain reaction (PCR) techniques to identify and screen for prevalence of parasites has a huge potential. It allows for the detection and identification of low-intensity infections, but its high sensitivity and technical setup may also induce problems. Here, we report a cautionary note regarding misleading amplification of avian malaria species (Haemoproteus and Plasmodium) during Leucocytozoon spp. detection. We used a previously described nested PCR method for the molecular detection of avian malaria and Leucocytozoon spp. In the first step of the PCR protocol, these parasites are detected simultaneously; in the second PCR, Haemoproteus and Plasmodium spp. are separated from Leucocytozoon spp. However, in certain cases when a bird was infected with avian malaria, we obtained a slightly longer PCR product during the detection of Leucocytozoon spp. Our data imply that these “false” Leucocytozoon fragments are the consequences of strong amplification of certain malaria lineages in the first PCR, which can also be detected after the second PCR amplification that is specific to Leucocytozoon spp. parasites. Because these “false” Leucocytozoon fragments are slightly longer than the normal Leucocytozoon fragments, we suggest the use of well-separating agarose gels, several positive controls, and molecular standards to facilitate their separation. If one obtains a fragment that differs in length from the one expected for Leucocytozoon spp., sequencing is essential. More generally, in order to limit this type of problem with nested PCR protocols, we suggest that the first and the second primer pair be chosen so that they have different annealing temperatures. (Less)
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author
organization
publishing date
type
Contribution to journal
publication status
published
subject
in
Journal of Parasitology
volume
94
issue
2
pages
562 - 564
publisher
American Society of Parasitologists
external identifiers
  • wos:000256207200038
  • scopus:43949143163
ISSN
0022-3395
DOI
10.1645/GE-1286.1
language
English
LU publication?
yes
id
303ddb5c-1daa-45c4-862b-dd8d21d20222 (old id 787157)
date added to LUP
2008-01-02 18:56:05
date last changed
2017-09-03 04:24:26
@article{303ddb5c-1daa-45c4-862b-dd8d21d20222,
  abstract     = {The use of new powerful nested polymerase chain reaction (PCR) techniques to identify and screen for prevalence of parasites has a huge potential. It allows for the detection and identification of low-intensity infections, but its high sensitivity and technical setup may also induce problems. Here, we report a cautionary note regarding misleading amplification of avian malaria species (Haemoproteus and Plasmodium) during Leucocytozoon spp. detection. We used a previously described nested PCR method for the molecular detection of avian malaria and Leucocytozoon spp. In the first step of the PCR protocol, these parasites are detected simultaneously; in the second PCR, Haemoproteus and Plasmodium spp. are separated from Leucocytozoon spp. However, in certain cases when a bird was infected with avian malaria, we obtained a slightly longer PCR product during the detection of Leucocytozoon spp. Our data imply that these “false” Leucocytozoon fragments are the consequences of strong amplification of certain malaria lineages in the first PCR, which can also be detected after the second PCR amplification that is specific to Leucocytozoon spp. parasites. Because these “false” Leucocytozoon fragments are slightly longer than the normal Leucocytozoon fragments, we suggest the use of well-separating agarose gels, several positive controls, and molecular standards to facilitate their separation. If one obtains a fragment that differs in length from the one expected for Leucocytozoon spp., sequencing is essential. More generally, in order to limit this type of problem with nested PCR protocols, we suggest that the first and the second primer pair be chosen so that they have different annealing temperatures.},
  author       = {Söllözi, Eszter and Hellgren, Olof and Hasselquist, Dennis},
  issn         = {0022-3395},
  language     = {eng},
  number       = {2},
  pages        = {562--564},
  publisher    = {American Society of Parasitologists},
  series       = {Journal of Parasitology},
  title        = {A cautionary note on the use of nested-PCR for parasite screening - an example from avian blood parasites},
  url          = {http://dx.doi.org/10.1645/GE-1286.1},
  volume       = {94},
  year         = {2008},
}