PCR-based detection of plasmodium in Anopheles mosquitoes: a comparison of a new high-throughput assay with existing methods
Author(s)
Type
Journal Article
Abstract
Background: Detection of the four malaria-causing Plasmodium species (Plasmodium falciparum, Plasmodium vivax,
Plasmodium ovale and Plasmodium malariae) within their mosquito hosts is an essential component of vector
control programmes. Several PCR protocols have been developed for this purpose. Many of these methods, while
sensitive, require multiple PCR reactions to detect and discriminate all four Plasmodium species. In this study a
new high-throughput assay was developed and compared with three previously described PCR techniques.
Methods: A new assay based on TaqMan SNP genotyping was developed to detect all four Plasmodium species
and discriminate P. falciparum from P. vivax, P. ovale and P. malariae. The sensitivity and the specificity of the new
assay was compared to three alternative PCR approaches and to microscopic dissection of salivary glands in a
blind trial of 96 single insect samples that included artificially infected Anopheles stephensi mosquitoes. The
performance of the assays was then compared using more than 450 field-collected specimens that had been
stored on silica gel, in ethanol or in isopropanol.
Results: The TaqMan assay was found to be highly specific when using Plasmodium genomic DNA as template.
Tests of analytical sensitivity and the results of the blind trial showed the TaqMan assay to be the most sensitive
of the four methods followed by the 'gold standard' nested PCR approach and the results generated using these
two methods were in good concordance. The sensitivity of the other two methods and their agreement with the
nested PCR and TaqMan approaches varied considerably. In trials using field collected specimens two of the
methods (including the nested protocol) showed a high degree of non-specific amplification when using DNA
derived from mosquitoes stored in ethanol or isopropanol. The TaqMan method appeared unaffected when using
the same samples.
Conclusion: This study describes a new high-throughput TaqMan assay that very effectively detects the four
Plasmodium species that cause malaria in humans and discriminates the most deadly species, P. falciparum, from
the others. This method is at least as sensitive and specific as the gold standard nested PCR approach and because
it has no requirement for post-PCR processing is cheaper, simpler and more rapid to run. In addition this method
is not inhibited by the storage of mosquito specimens by drying or in ethanol or isopropanol.
Plasmodium ovale and Plasmodium malariae) within their mosquito hosts is an essential component of vector
control programmes. Several PCR protocols have been developed for this purpose. Many of these methods, while
sensitive, require multiple PCR reactions to detect and discriminate all four Plasmodium species. In this study a
new high-throughput assay was developed and compared with three previously described PCR techniques.
Methods: A new assay based on TaqMan SNP genotyping was developed to detect all four Plasmodium species
and discriminate P. falciparum from P. vivax, P. ovale and P. malariae. The sensitivity and the specificity of the new
assay was compared to three alternative PCR approaches and to microscopic dissection of salivary glands in a
blind trial of 96 single insect samples that included artificially infected Anopheles stephensi mosquitoes. The
performance of the assays was then compared using more than 450 field-collected specimens that had been
stored on silica gel, in ethanol or in isopropanol.
Results: The TaqMan assay was found to be highly specific when using Plasmodium genomic DNA as template.
Tests of analytical sensitivity and the results of the blind trial showed the TaqMan assay to be the most sensitive
of the four methods followed by the 'gold standard' nested PCR approach and the results generated using these
two methods were in good concordance. The sensitivity of the other two methods and their agreement with the
nested PCR and TaqMan approaches varied considerably. In trials using field collected specimens two of the
methods (including the nested protocol) showed a high degree of non-specific amplification when using DNA
derived from mosquitoes stored in ethanol or isopropanol. The TaqMan method appeared unaffected when using
the same samples.
Conclusion: This study describes a new high-throughput TaqMan assay that very effectively detects the four
Plasmodium species that cause malaria in humans and discriminates the most deadly species, P. falciparum, from
the others. This method is at least as sensitive and specific as the gold standard nested PCR approach and because
it has no requirement for post-PCR processing is cheaper, simpler and more rapid to run. In addition this method
is not inhibited by the storage of mosquito specimens by drying or in ethanol or isopropanol.
Date Issued
2008-09-15
Date Acceptance
2008-09-15
Citation
Malaria Journal, 2008, 7
ISSN
1475-2875
Publisher
BioMed Central
Journal / Book Title
Malaria Journal
Volume
7
Copyright Statement
© 2008 Bass et al.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),
which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Identifier
http://gateway.webofknowledge.com/gateway/Gateway.cgi?GWVersion=2&SrcApp=PARTNER_APP&SrcAuth=LinksAMR&KeyUT=WOS:000260012400002&DestLinkType=FullRecord&DestApp=ALL_WOS&UsrCustomerID=1ba7043ffcc86c417c072aa74d649202
Subjects
Science & Technology
Life Sciences & Biomedicine
Infectious Diseases
Parasitology
Tropical Medicine
PARASITOLOGY
TROPICAL MEDICINE
POLYMERASE-CHAIN-REACTION
LINKED-IMMUNOSORBENT-ASSAY
CIRCUMSPOROZOITE PROTEIN
FALCIPARUM GAMETOCYTES
MALARIA PARASITES
FIELD SAMPLES
IDENTIFICATION
GAMBIAE
INFECTIVITY
CULICIDAE
Animals
Anopheles
DNA, Protozoan
Humans
Microscopy
Plasmodium
Polymerase Chain Reaction
Polymorphism, Single Nucleotide
Salivary Glands
Sensitivity and Specificity
Medical Microbiology
Publication Status
Published
Article Number
177