The Centre for Research in Infectious Diseases (CRID) situated in Yaoundé – Cameroon provides comfortable, safe and well equipped laboratories to conduct various research in the domains of Medical Entomology, Parasitology and Virology. Our molecular biology platform is conducive to perform experiments including;
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DNA and RNA extraction
Using different protocols, we are able to extract DNA or RNA from living cells which are later used as templates for amplifications of genes of interest by PCR. The platform is well equipped with material necessary for DNA/RNA extraction such as centrifuges (simple and refrigerated), water baths, spinner, vortex, grinder, Nanodrop for quantification of nucleic acids after extraction.
DNA amplification and genotyping
The platform for DNA amplification and genotyping is equipped with several thermocyclers, electrophoresis generators, electrophoresis tanks and electrophoresis documentation system
Various genotyping protocols are performed allowing anopheles species identification, detection of the Plasmodium parasites in the mosquito, determination of genotypes at loci conferring the ability to mosquitoes to survive after exposition to insecticides. Current methods of genotyping used at CRID include:
- Restriction fragment length polymorphism (RFLP-PCR),;
- Allele-specific-PCR (AS-PCR);
- TaqMan genotyping method;
Quantitative PCR and Real time PCR
This method is used to study gene expression vectors. This technique is used at CRID to:
- Validate the results of differential expression of genes implicated in metabolic resistance in mosquitoes. These results are obtained from Microarray and RNA-sequencing techniques;
- Assess the expression profiling of some candidate genes involved in metabolic resistance;
- Assess differential level of expression of key genes involved in immune response of Anopheles to Plasmodium infection;
- Assess the expression profiling in resistant mosquitoes of genes encoding for salivary proteins involved in blood feeding process and in Plasmodium invasion in the salivary glands;
- Single nucleotide polymorphism (SNP) analysis and allele discrimination;
- Detecting and genotyping arboviruses infection in humans, animals and mosquitoes;
CRID disposes of two Real time PCR machines connected to computers with every necessary reagent and consumables to perform this technique.
At CRID, we perform molecular cloning by following a set of protocols including;
- Insertion of target genes in plasmids for sequencing and protein expression;
- Preparation of chemo competent cells for bacteria transformation;
- Protein expression of mosquito detoxification genes like cytochrome P450s, Glutathione S transferase and carboxylesterases;
- Luciferase promoter assay where the promoter of genes are clone upstream luciferase reporter gene to determine the strength of the promoter;
- Protein profiling using 2 dimension gels electrophoresis;
For this platform, CRID has two incubators; simple and shaking incubator a laminar flow hood, an autoclave and all the necessary reagents and consumables to carry out this technique.
Double-stranded RNA-mediated interference (RNAi) is a technology, which allows us to silence the expression of gene of interest in a variety of organisms. It is a simple and rapid method that consists of knocking down a gene via sequence-specific manner by injecting synthesised double strand RNA in the cell of the organism. At CRID RNAi is mainly used to study the impact of candidate genes involved in insecticide resistance in mosquitoes. After the process of RNAi, if the level of susceptibility in mosquitoes injected is higher than that of the control group (non-injected mosquitoes), this validates the implication of the target gene (gene knock-down) in insecticide resistance.
Our lab also provides a platform to perform RNAi were we have:
- Nano injector for injection, a puller to make needles used;
- A CO2 equipment to anesthetise the organism during the procedure;
- qPCR machine for quantitative PCR to verify the gene knockdown;
- Thermo cycler for Semi-quantitative PCR ;
Microarray is one of the best tools available to perform gene expression profiling. This technique offers the possibility to compare the difference in gene expression between two mRNA samples. Our laboratory has;
– Nanodrop to verify quality and to quantify the RNA. Oven for hybridization and all the necessary reagents and consumables. The esteemed collaboration with the Liverpool School of Tropical Medicine (LSTM), allows us to complete the experiment in Liverpool with a bioanalyser, followed by scanning, normalisation and analysis;
Our HPLC facility enables us to perform routine identification; quantification and separation of analytes using a C18 column mounted on Agilent 1200 HPLC system with UV detector. We have established robust protocols using this chromatographic technique to support investigation in:
- Evaluating metabolites generated by metabolism of insecticide by detoxification enzymes over-expressed in resistant pest and disease vectors;
- Quality control analysis of Insecticide Treated Net (ITNs);
- Insecticide residues in soil, water and crops;
- Evaluating the Maximum Residual Limit (MRL) of insecticides in agricultural products;
ELISA (Enzyme-Linked Immuno Sorbent Assay)
ELISA CSP (circumsporozoites proteins) This immunological technique is used at CRID to determine mosquito infection status. CRID possesses the complete platform with all antibodies available and an Elisa Reader (Microplate Reader).
ELISA Blood Meal
It is the enzyme immunoassay, using blood-fed vectors such as mosquitoes to determine the source of blood meals using polyclonal antibodies from humans, beef, sheep, chickens, horses, pigs, and dogs. This technique gives us the feeding preferences of mosquitoes
ELISA is used to assess past or acute infection of arboviral related diseases like dengue, Zika, yellow fever, chikungunya and rift valley fever by detecting immunoglobulin G (IgG) or IgM in animal and human sera.
In the context of malaria elimination and eradication, it is important to evaluate the vectorial competence of the major malaria vector through experimental infections. The experimental infections assays are performed in our laboratory under intensely controlled conditions to quantify the transmission modulating effect of naturally acquired immune responses against Plasmodium spp. Studies to identify different immune genes that play an important role in reducing or eliminating Plasmodium in mosquitoes are receiving increased attention. Despite the defence mechanism of mosquitoes, some plasmodium manage to escape these defence mechanisms and continue their development to the sporozoite stage.
Experimental infection consists of taking blood from patients with gametocytes. This blood is given to the mosquitoes through the standard membrane-feeding assay. After 7 days’ post-infection, midguts will be dissected to evaluate the plasmodial infection burden by counting the number of oocysts and after 14 days post-infection, we evaluate the plasmodial infection burden by detecting the sporozoites. Finally, the different groups of mosquitoes (infected and uninfected) allow us to know by transcriptional analysis which immune genes are expressed by both the mosquito and the parasite.