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Assessment of pfmdr 1 gene copy number by tandem competitive polymerase chain reaction
R Price1, G Robinson, A Brockman
1Division of Infectious Diseases, St. George's Hospital Medical School, London, UK.
Abstract:
The pfmdr 1 gene encodes a Plasmodium falciparum homologue of the human P-glycoprotein expressed on the surface of the parasite food vacuole. Variation in copy number and specific codon mutations of pfmdr 1 have been implicated in the development of parasite resistance to antimalarial drugs. We describe here the technique of Tandem-Competitive Polymerase Chain Reaction (TC-PCR), which allows accurate measurement of pfmdr 1 copy number in parasite DNA obtained directly from small quantities (100 microliters) of red blood cells. We reliably quantified pfmdr1 in previously well characterised strains of Plasmodium falciparum with differing pfmdr1 gene copy numbers using starting amounts of between 3,000 and 40,000 gene copies. We then used TC-PCR to determine pfmdr1 gene copy number in field specimens of venous blood taken from 10 patients with malaria contracted along the Thai-Burmese border. In this region of high grade parasite resistance to mefloquine greater than 70% of samples had a copy number greater than 1 of pfmdr1 determined with a repeatability coefficient of 0.58.
Insights
A new Tandem-Competitive Polymerase Chain Reaction (TC-PCR) method accurately measures Plasmodium falciparum pfmdr1 gene copy number. This technique aids in understanding antimalarial drug resistance in malaria patients.
Area of Science:
- Molecular biology
- Parasitology
- Genetics
Background:
- The pfmdr1 gene in Plasmodium falciparum is crucial for antimalarial drug resistance.
- Variations in pfmdr1 copy number and mutations are linked to reduced drug efficacy.
Purpose of the Study:
- To develop and validate a precise method for quantifying pfmdr1 gene copy number.
- To assess pfmdr1 copy number in clinical malaria samples from a drug-resistant region.
Main Methods:
- Tandem-Competitive Polymerase Chain Reaction (TC-PCR) was developed for accurate pfmdr1 copy number measurement.
- The method was validated using characterized Plasmodium falciparum strains.
- TC-PCR was applied to DNA from patient blood samples.
Main Results:
- TC-PCR reliably quantified pfmdr1 in Plasmodium falciparum strains with known copy numbers.
- In malaria patients from the Thai-Burmese border, over 70% showed pfmdr1 copy number greater than 1.
- The method demonstrated high repeatability (coefficient of 0.58).
Conclusions:
- TC-PCR is an effective technique for measuring pfmdr1 gene copy number from small blood volumes.
- Elevated pfmdr1 copy number is prevalent in malaria parasites from a mefloquine-resistant area.
- This method can aid in monitoring antimalarial drug resistance patterns.