Detection of plasmodium parasites in field samples using conventional PCR

Abstract

Malaria is currently the dealing cause of death among children in Uganda. It’s transmitted to humans through bites of infected female anopheles mosquito. Currently, malaria case detection depends heavily on microscopy and/or Rapid Diagnosis Tests (RDTs). However, both methods have a tendency of missing to detect infections especially when parasite densities are low (<10 parasites/μl). In this study microscopy and RDT negative samples were analysed for plasmodium parasites using conventional PCR. Blood samples were collected from Katanga, Kampala District in EDTA vacutainers. Genomic DNA was extracted from the blood samples using STE buffer method that involved use of reagents: sodium chloride Tris EDTA Buffer (STE buffer), chloroform- isoamyl alcohol, 3M sodium acetate, both 95% and 70% ethanol. STE buffer (250µl) was added to blood sample (200µl) followed by incubation at 650C. 24µl: 1µl chloroform: isoamyl alcohol (500µl) were added followed by centrifugation at 13000 rpm. Phase separation was done and 400µl of the aqueous layer were picked off into a new micro centrifuge tube. The DNA was precipitated using 95% ethanol and 3M sodium acetate and washed using 70% ethanol.as described well in the methodology. This was followed by DNA amplification using plasmodium specific primers: rPLAS (5′ TTAAAATTGTTGCAGTTAAAACG-3′) and fPLAS (5′-CCTGTTGTTGCCTTAAACTT- 3′) at annealing temperature of 500C for 30 cycles. A total reaction volume of 10µl was used. The results from this study showed that 66% (39/59) were positive malaria cases and 34 % (20/59) were negative malaria cases. PCR also was able to detect 66% malaria cases that were missed by the microscopy and RDT further confirming PCR as a very important method in malaria diagnosis. The sample size used was small and could not be used to conclusively confirm the results from this study, hence a bigger research needs to be done. Reagents used were not enough to permit more sample analyses, hence with availability of more reagents, a bigger picture of the research can be achieved.