The overall goal of this procedure is to extract malaria, parasite, and mosquito DNA from the midgut and salivary gland sections of anomalies, specimens. This is accomplished by first cutting the mosquito exactly at the joint between the abdomen and the thorax, thus separating the midgut from the salivary gland section. Next, each section is ground separately in deionized water.
Then the tissue suspension is lyed and washed and the cell pellet is recovered. Finally, the cell pellet is reconstituted and boiled, and the DNA is extracted. Ultimately, results can be obtained that show high quality mosquito and malaria parasite, DNA, suitable for use as template to identify anomalies, vector species, and genotype malaria infections through PCR and allele specific restriction enzyme digestion.
So the main advantage of this very simple technique over existing ones like organic extraction and sorting out methods is that it uses very few safe and readily available reagents and much less processing time as well. To begin dissecting the mosquito, place it on a small piece of paraform and mount it on a dissecting microscope. Cover it in a drop of deionized water.
To soften the tissue in size, the mosquito carcass precisely at the joint between the thorax and abdomen. Thus nicking off the head and thorax from the abdominal section. Transfer each dissected mosquito section into a clean autoclave, 1.5 milliliter micro centrifuge tube pre-labeled with mosquito ID number and body section discard the para film and using a 70%alcohol moistened Kim wipe carefully wipe the microscope stage before processing to the next mosquito to extract mosquito DNA first pipette 20 microliters of deionized water into the sample tube.
And use the pipette tip to grind the submerged mosquito section into a uniform suspension. Add 100 microliters of an autoclave, one XPBS 1%saponin solution to the sample homogenate and gently vortex to mix. Incubate the sample at room temperature for 20 minutes.
Next centrifuge at 20, 000 G for two minutes, and discard the supernatant. Resuspend the pellet in 100 microliters one XPBS. Then repeat the spin.
Add 75 microliters of sterile deionized water and 25 microliters of 20%weight per volume. Cellex 100 resin suspension in deionized water and gently vortex to resuspend the pellet using a sterile 23 gauge hypodermic needle flamed in bunsen burner. Pierce a hole in the lid of the sample tube.
Boil the sample suspension in a heating block for 10 minutes. Centrifuge at 20, 000 G for one minute, and transfer the DNA solution into a pre-labeled storage vial for use as the template in PCR reactions to check the quality of the extracted DNA, amplify a region of the arthropod mi mitochondrial nicotinamide adenine dinucleotide dehydrogenase subunit four gene that is expected to produce a 400 base pair fragment. Next to differentiate between Ananais Gambia species, amplify a region flanking SNPs in the intergenic spacer region that is expected to produce sizes of 390 base pairs for Ananais Gambia Sens Stricto, and 315 base pairs for ananais api.
Ansis then to genotype p falciparum anti folate drug resistance polymorphisms. Perform nested PCR to amplify the region flanking amino acid codons 16 51 59, 108, and 164. In the parasites dihydro folate reductase gene carry out allele specific digestion of the amplicon to detect anti folate drug resistance.
Associated polymorphisms. Check the reactions on a 2%AROS gel exam. Examples of results from PCR assays for mosquito DNA extract quality analis RABIS molecular identification, and p falciparum genotyping show that the simplified Kelex method yields similar results to the standard salting out protocol despite fewer steps and less than a quarter of the time with comparable DNA quality in the respective extracts, it is not surprising that sample positivity rates with respect to anomalies, Gambia sibling species, as well as parasite infection rates were not statistically different based on McNear's chi square test.
The addition of BSA to reaction mixtures results in a general increase of PCR positives due to removal of PCR inhibitors for both the simplified kelex and regular salting out protocol. However, this increase was not statistically significant except for DNA quality on kelex extracts. Allele specific restriction enzyme digestion of the p falciparum amplicon enables the genotyping of midgut and salivary gland malaria infections for drug resistance alleles Once mastered.
This technique should be able to be performed in about 37 minutes if performed properly.
