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, SNP)^13-18, (3) parasite detection PCR^19-23, and (4) screening for host-specific DNA in mosquito midguts^24,25.

We developed a multi-detection assay (MDA) that combines all these assays into a single multiplex reaction with the goal of analyzing epidemiologically important characteristics of malaria vectors in Africa. The MDA assay includes multiple markers for detecting (1) species (A. arabiensis, A. gambiae, A. coluzzii, or other (none of the three)), (2) insecticide resistance represented in kdr SNPs (both L1014Fand L1014S), (3) presence of two major malaria parasites, Plasmodium falciparum and P. vivax, and (4) blood source from one avian and six mammalian hosts.

Traditionally, these assays were conducted in separate polymerase chain reactions. When all these assays are done using a conventional PCR platform, it requires conducting 8-10 PCR reaction assays and accompanying gel electrophoresis steps. Each individual PCR reaction from preparation to documenting results takes 4-5 hr, while the MDA method presented here takes about 5 hr in totality. This is equivalent to a 90% savings in labor costs alone. The MDA presented here costs $5 per sample to genotype all 33 SNPs. This is considerably less expensive than the single agarose gel-based assays, which costs about $1.50 per sample. Assays detecting all the characteristics covered by the MDA would require a minimum of 8-10 separate agarose gel-based assays at a cost of $12-15 per sample. Moreover, the MDA greatly reduces the chance of generating a false positive or negative by utilizing at least three markers for each parasite or host source detection.

The platform we utilized is not limited to malaria vectors but can be used in a wide variety of applications such as medicine, veterinary medicine, and basic biology^26-28. In-depth association studies or population genetics studies involving a large (in order of 100s) number of samples require cost-effective assays screening for multiple markers simultaneously. Most studies that utilize two or more separate PCR assays could implement the MDA for quicker results at a lower cost.

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 at 2,000 x g.
3. Dispense 3 μl of the PCR cocktail into each well of a non-skirted 96 well plate. A repeater pipette can save time for this process.
4. Add 2 μl of the appropriate genomic DNA of mosquitoes in each well.
   NOTE: Process of obtaining genomic DNA from mosquito tissues (head/thorax, abdomen or whole body) is described in other literatures^29-32. Choose an appropriate DNA extraction protocol to differentiate those with Plasmodium in the blood (abdomen) from infective mosquitoes (Plasmodium in the head/thorax). We typically use DNA extracted from body parts (head/thorax or abdomen) of mosquitoes, so DNA concentration varies from 0.05-2ng/μl. Although the total DNA input is lower than the manufacturer’s recommendation (5-10ng/rxn), we typically get robust SNP calls from 98% of DNA samples without whole genome amplification.
5. Seal the plate, gently mix or vortex, and centrifuge for 1 min at 370 x g.
6. Use the following thermocycler protocol to carry out PCR amplification: (1) 94°C for 2 min, (2) 94°C for 30 sec, (3) 56°C for 30 sec, (4) 72°C for 1 min, (5) repeat step (2)-(4) for 45 cycles, (6) 72°C for 1 min, and 4°C hold.

2. SAP Treatment

1. Prepare the SAP enzyme solution in a 96 well plate (See Table 2). Process 96 samples in a plate. The volume for 96 reactions includes an overhang of 20%.
2. Vortex the tube of SAP enzyme solution lightly and centrifuge for 30 sec at 2,000 x g. Dispense 2 μl of the SAP enzyme solution into each well. Again, a repeater pipette can save time for this process.
3. Seal the plate, gently mix or vortex, and centrifuge for 1 min at 370 x g. Incubate the plate as follows: (1) 37°C for 40 min, (2) 85°C for 5 min to inactivate the enzyme, (3) 4°C hold. At completion of this step, store the plate at -20°C before continuing. Process the stored plate within 2 weeks.

3. SNP Extension

1. If the sample plate is frozen after SAP treatment, allow it to thaw to RT before proceeding.
2. Prepare the extension primer mix (Supplemental Table S2). Each SNP has one extension primer. Keep the stock concentration of each extension primer at 500 μM. Optionally, aliquot for 100-200 reactions per tube and store at -20°C.
3. Prepare extension mix as described in Table 3. The 96 reaction volume includes an overhang of 20%.
4. Vortex the tube of extension cocktail lightly and centrifuge for 30 sec at 2,000 x g. Dispense 2 μl of the extension cocktail into each well. Again, a repeater pipette can save time for this process.
5. Seal the plate, gently mix or vortex, and centrifuge for 1 min at 370 x g. Thermocycle the plate as shown in Figure 1. At this point, proceed to mass spectrometry or store the plate at -20°C up to 2 weeks.

4. Conditioning the SNP Extension Product to Optimize Mass Spectrometry Analysis

1. If the sample plate is frozen after SNP extension, allow it to thaw to RT before proceeding.
2. Transfer 15mg of resin from its container onto the dimple plate using an elongated spoon. Use the scraper to spread resin into the wells of the dimple plate. Sweep the scraper from side to side across the dimple plate to spread the resin. Make sure there is resin in each well.
3. Scrape excess resin off the dimple plate using the scraper. Let the resin stand in the dimple plate for at least 20 min. While letting the resin stand in the dimple plate, add 41 μl of HPLC grade water to each well of the sample plate.
4. Gently place the sample plate upside-down, onto the dimple plate. Make sure the sample plate resets against the small rounded post of the dimple plate, which aligns the wells in the sample plate with the wells in the dimple plate.
5. Holding the sample plate and the dimple plate together, gently flip them over so the resin falls out of the dimple plate into the wells of the sample plate. Tap the dimple plate so the resin falls out into the sample plate. Make sure all the resin in the dimple plate falls out into the sample plate wells. Centrifuge the plate at 3,200 x g for 30 sec.
6. Rotate the sample plate on a rotator for 5 min at RT. Rotator must rotate the sample plate 360° about the long axis.
7. Centrifuge the sample plate at 3,200 x g for 5 min. After this step, store the sample plate at -20°C up to 2 weeks before proceeding to the next step.

5. MALDI-TOF Mass Spectrometry

1. Transfer the conditioned SNP extension product onto a bioarray such as SpectroCHIP as per manufacturer’s instructions. We use MassArray Nanodispenser for this step.
2. Once the transfer is complete, define how assays and plates are to be set up in the assay database. Name the assay and plates such that each plate has unique identifier that links to specific assay design (Supplemental Table S3).
3. After assay and plate have been defined, acquire spectra using a mass spectrometer.

6. Data Analysis

1. Perform data analysis using a real-time PCR analysis data interpretation software such as Typer Analyzer or TyperViewer (Sequenom). Obtain data in .xml format which contains the sample layout, mass spectrogram for each well, the marker information including name, expected mass for each variant of the corresponding SNP, and any error or warning messages associated with each reaction. See Figure 2 for the resulting mass spectrogram.
2. Carry out SNP genotype clustering analysis by setting the detection threshold (magnitude or signal to noise ratio) and tolerance/variance allowed for each genotype. Figure 3 shows an example of a 04707-KDR#2 SNP genotype clusters.
3. Export the resulting SNP genotype calls into a spreadsheet program.

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.