A Multi-detection Assay for Malaria Transmitting Mosquitoes

Summary

Malaria transmitting mosquitoes have a number of epidemiologically important characteristics that can only be detected using molecular techniques. Utilizing a MALDI-TOF based SNP genotyping platform, we developed an assay for simultaneously detecting multiple key traits (species, insecticide resistance, parasite infection and host choice) of malaria vectors.

Abstract

The Anopheles gambiae species complex includes the major malaria transmitting mosquitoes in Africa. Because these species are of such medical importance, several traits are typically characterized using molecular assays to aid in epidemiological studies. These traits include species identification, insecticide resistance, parasite infection status, and host preference. Since populations of the Anopheles gambiae complex are morphologically indistinguishable, a polymerase chain reaction (PCR) is traditionally used to identify species. Once the species is known, several downstream assays are routinely performed to elucidate further characteristics. For instance, mutations known as KDR in a para gene confer resistance against DDT and pyrethroid insecticides. Additionally, enzyme-linked immunosorbent assays (ELISAs) or Plasmodium parasite DNA detection PCR assays are used to detect parasites present in mosquito tissues. Lastly, a combination of PCR and restriction enzyme digests can be used to elucidate host preference (e.g., human vs. animal blood) by screening the mosquito bloodmeal for host-specific DNA. We have developed a multi-detection assay (MDA) that combines all of the aforementioned assays into a single multiplex reaction genotyping 33SNPs for 96 or 384 samples at a time. Because the MDA includes multiple markers for species, Plasmodium detection, and host blood identification, the likelihood of generating false positives or negatives is greatly reduced from previous assays that include only one marker per trait. This robust and simple assay can detect these key mosquito traits cost-effectively and in a fraction of the time of existing assays.

Introduction

Anopheles arabiensis, Anopheles coluzzii and Anopheles gambiae are the major vectors responsible for malaria transmission in Africa¹. These three species are morphologically indistinguishable² and can only be distinguished by molecular assays^3-9. In addition, there are many downstream assays routinely conducted to aid epidemiological and population genetics studies. These include (1) a genotyping assay for speciation islands^10-12, (2) a genotyping assay recognizing non-synonymous SNPs in the 1014^th amino acid codon position of para gene (the knock-down resistance, or kdr,

Protocol

1. PCR Amplification

1. Mix all the PCR primers (See supplemental Table S1) in a 1.5ml microtube. Each SNP has two primers (forward and reverse). Ensure that the stock concentration of each PCR primer is kept at 100 μM. Make enough primer mix for 500-1,000 reactions to reduce pipetting error and time at the bench (See supplemental Table S2). If desired, prepare aliquots for 100-200 reactions per tube and store at -20°C.
2. Prepare PCR cocktail as described in the manufacturer’s protocol (Table 1). The volume for 96 reactions includes an overhang of 20%. Vortex the tube containing the PCR cocktail lightly and centrifuge for 30 sec a....

Results

Species identification:

The following 5 SNPs together identify three species (A. arabiensis, A. coluzzii and A. gambiae) (Table 4). If a sample is not one of the three species, the three SNPs (01073-213, 04679-157 and 10313-052) fail to amplify.

kdr genotype for inferring insecticide resistance:

The 1014^th codon of the para voltage-gated sodium channel cor.......

Discussion

The MDA is composed of five major steps: PCR amplification, shrimp alkaline phosphatase (SAP) reaction, SNP extension, extension product conditioning, and matrix-assisted laser desorption/ionization – time of flight (MALDI-TOF) mass spectrometry^33-37. The first PCR amplification step amplifies DNA flanking each SNP so that enough template DNA will be available at the SNP extension step. The SAP reaction neutralizes unused dNTPs which can interfere with the following SNP extension step. SNP extension invo.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
MassARRAY Analyzer Compact	Sequenom	MT9	MALDI-TOF mass spectrometry for genomic applications to analyze nucleic acids.
MassARRAY Nanodispenser	Sequenom	RS1000	Transfers completed iPLEX reaction products to the SpectroCHIP
iPLEX Gold Genotyping Reagent Set	Sequenom	10158	Reagents used for iPLEX assay including SAP kit.