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




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Malaria Journal

, 16:230

First Online: 31 May 2017Received: 25 January 2017Accepted: 26 May 2017DOI: 10.1186-s12936-017-1881-1

Cite this article as: Echeverry, D.F., Deason, N.A., Makuru, V. et al. Malar J 2017 16: 230. doi:10.1186-s12936-017-1881-1

Abstract

BackgroundMolecular 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.

MethodsIn 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.

ResultsThe 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.

ConclusionsThis 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.

KeywordsMalaria Plasmodium Diagnosis Sporozoite Anopheles 18s-rRNA Cytochrome oxidase I Solomon Islands Vectors DNA barcoding AbbreviationsCSP-ELISAcircumsporozoite protein-enzyme-linked immunosorbent assay

COX-Icytochrome oxidase I

18s-rRNA18 small subunit ribosomal RNA

BEI Resourcesthe biological and emerging infections resources program

minminute

secsecond

µLmicrolitre

pgpicogram

ngnanogram





Author: Diego F. Echeverry - Nicholas A. Deason - Victoria Makuru - Jenna Davidson - Honglin Xiao - Julie Niedbalski - Xiaoyu Yu

Source: https://link.springer.com/







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