Hi all,
I have a following query. Please help
From a given sample (malaria positive sample), following an extraction by Qiamp blood Kit, Does the concentration of DNA yield estimated by spectrophotometer represent how many of the parasites were present in the sample ? How many of copy number of target genes each form of malaria parasite carry?lets say 18s rRNA
How would you determine the parasitemia in Malaria positive patients? My point is the number of copies of target genes would not represent the parasitemia.
i am very new to this subject matter. Please suggest
Thanking you in an advance
Hello...
I am not sure to understand your question...
what is the malaria-positive sample? Because if you performed DNA purification, you won't have only malaria DNA, but also DNAf rom the host...You should be aware that the yield of extraction kit is not 100%, so it seems a bad idea, knowing the size of the genome of Plasmodium, to calculate the number of genomes = number of parasites) with the concentration...
If you want to know how much DNA for 100, 1000 parasites, you may need to isolate 100, 1000 parasites in blood, then purified DNA (can you isolate cells by microscopy?) This will be your reference, then do a qPCR to compare it to positive sample.
I don't understand your question about copy number and form of malaria, sorry.
Thank you very much for your reply.
You are right. From the given blood sample (Malaria positive sample- is the sample in which malaria parasite is confirmed present), I will not only have parasitic DNA but also a human DNA. The best way to do it i guess is either with purifying the parasites from in vitro or removing human DNA from the sample (both are not available in our present situations) to know the exact DNA yield by spec. However, Since we do not have quantitative standards available (for copy number of target genes), can we analyze the DNA in a positive control (supplied by the manufacturer) based on Spec quantification and prepare series of dilution of the control and compare with the unknown sample? can we get the copy number from known DNA in the positive control?
my next question was: How would you determine parasitemia in malaria infected patients by qPCR? Thank you
The parasitemia could be estimated by extrapolating from a standard curve generated by extracting nucleic acid from in vitro P. falciparum culture whose parasite density is known. In in vitro culture system , human DNA contamination is minimal and therefore the resulting absorbance is mostly due to parasites.
The number of parasites in the blood and the number of target genes in the parasite is different. The number of target genes may be one, two or more than two in a single parasite depending on the nature of genes you target. To know the number of parasites in the blood you have to be sure that your DNA isolation mechanism isolates only the parasites DNA in the blood 100% efficiently.
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