The detection of Plasmodium falciparum and P-vivax in DNA-extracted blood samples using polymerase chain reaction

Seventeen pairs of published primer sets were compared for their relative s ensitivity to detect malaria DNA extracted from blood samples, which were o btained from Pakistani patients suffering from malaria. The primer sets inv estigated consisted of. (i) 9 pairs of direct primers and 3 sets of nested primers for detecting Plasmodium falciparum, (ii) 2 pairs of direct primers and 2 sets of nested primers for detecting P. vivax, and (iii) 1 set of mu ltiplex primers for detecting both P. falciparum and P. vivax, simultaneous ly. After a miniscreen of 9 DNA-extracted blood samples using the 17 primer sets stated above, 5 primer sets were short-listed (based on their superio r sensitivity) and used for a maxi-screen of DNA extracted from 126 microsc opy-positive blood samples from Pakistan, with the following results. (i) F or the detection of P. falciparum, the direct primer pair 'PF1+PF2' gave a sensitivity of 95% and the nested primer set 'RIT405+RIT406/RIT371+RIT372' gave a sensitivity of 97%. (ii) For the detection of P. vivax, the direct p rimer pair 'Forward+Reverse' and the nested primer set 'PLF+UNR/PLF+VIR' bo th gave a sensitivity of 94%. (iii) The nested multiplex primer set 'rPLU5rPLU6/rFAL1+rFAL2+rVIV1+rVIV2' gave a sensitivity of 97% and 96% for P. fal ciparum and P. vivax, respectively. It was concluded that the nested multip lex primer set was the most optimal primer set to use for the detection of malaria DNA extracted from blood samples. Furthermore, the nested multiplex primer set has the advantage of simultaneously detecting and differentiati ng between P. vivax and P. falciparum.