Fast and robust single PCR for Plasmodium sporozoite detection in mosquitoes using the cytochrome oxidase I gene

Echeverry, Diego F., Deason, Nicholas A., Makuru, Victoria, Davidson, Jenna, Xiao, Honglin, Niedbalski, Julie, Yu, Xiaoyu, Stevenson, Jennifer C., Bugoro, Hugo, Aparaimo, Allan, Reuben, Hedrick, Cooper, Robert, Burkot, Thomas R., Russell, Tanya L., Collins, Frank H., and Lobo, Neil F. (2017) Fast and robust single PCR for Plasmodium sporozoite detection in mosquitoes using the cytochrome oxidase I gene. Malaria Journal, 16. 230. pp. 1-8.

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Background: Molecular tools for detecting malaria-infected mosquitoes with improved practicality, sensitivity and specificity, and high-throughput are required. A common PCR technique used to detect mosquitoes infected with Plasmodium spp. is a nested PCR assay based on the 18s-rRNA gene. However, this technique has several technical limitations, is laborious and time consuming.

Methods: In this study, a PCR-based on the Plasmodium cytochrome oxidase I (COX-I) gene was compared with the 18s-rRNA nested PCR using serial dilutions (330–0.0012 pg) of DNA from Plasmodium vivax, Plasmodium falciparum and Plasmodium knowlesi and with DNA from 48 positive and negative Kenyan mosquitoes (previously detected by using both ELISA and PCR). This assay for Plasmodium spp. DNA detection using the fast COX-I PCR assay was then performed individually on 2122 field collected mosquitoes (from the Solomon Islands) in which DNA was extracted from head and thorax.

Results: The fast COX-I PCR assay took 1 h to run and consistently detected as low as to 0.043 pg of parasite DNA (equivalent to two parasites) in a single PCR, while analyses with the 18s-rRNA nested PCR required 4 h to complete with a consistent detection threshold of 1.5 pg of DNA. Both assays produced concordant results when applied to the 48 Kenyan control samples with known Plasmodium spp. infection status. The fast COX-I PCR identified 23/2122 Plasmodium-infected mosquitoes from the Solomon Islands.

Conclusions: This new COX-I PCR adapted for a single PCR reaction is a faster, simpler, cheaper, more sensitive technique amenable to high-throughput analyses for Plasmodium DNA detection in mosquitoes and is comparable to the 18s-rRNA nested PCR. The improved sensitivity seen with the fast COX-I PCR will improve the accuracy of mosquito infection rate determination.

Item ID: 49196
Item Type: Article (Research - C1)
ISSN: 1475-2875
Keywords: malaria, Plasmodium, diagnosis, sporozoite, Anopheles, 18s-rRNA, cytochrome oxidase I, Solomon Islands, vectors, DNA barcoding
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© The Author(s) 2017. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (, which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver ( applies to the data made available in this article, unless otherwise stated.

Funders: Gates Foundation Malaria Transmission Consortium (GF MTC), International Center of Excellence in Malaria Research in the Southwest Pacific (ICEMRSP)
Projects and Grants: GF MTC Grant ID 451, ICEMRSP U19AI089
Date Deposited: 28 Jun 2017 00:02
FoR Codes: 32 BIOMEDICAL AND CLINICAL SCIENCES > 3207 Medical microbiology > 320704 Medical parasitology @ 100%
SEO Codes: 92 HEALTH > 9204 Public Health (excl. Specific Population Health) > 920499 Public Health (excl. Specific Population Health) not elsewhere classified @ 80%
96 ENVIRONMENT > 9604 Control of Pests, Diseases and Exotic Species > 960405 Control of Pests, Diseases and Exotic Species at Regional or Larger Scales @ 20%
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