Immunomagnetic purification to facilitate DNA diagnosis of Plasmodium falciparum

Abstract

The detection of pathogens by polymerase chain reaction (PCR) in clinical samples, such as blood, urine, or feces, requires initial sample preparation to remove polymerase inhibitors and to concentrate the target DNA. Here we show for the first time that immunomagnetic separation can be used to recover pathogens from whole blood and then used for PCR analysis. With antibodies to the merozoite surface protein (MSP1), the malaria-causing parasite Plasmodium falciparum was purified and concentrated from clinical samples. The recovered parasites were used directly for in vitro DNA amplification. The PCR product was subsequently analyzed by a colorimetric 96-well microtiter plate assay. The results from examining 117 patients attending a clinic in the Borai district, Thailand, demonstrate that the combined method with immunomagnetic separation followed by PCR increases the group of positively diagnosed patients compared with microscopic examination of stained blood films. Analysis of 1 microliter of whole blood resulted in a 12% (14 of 117) increase in positively diagnosed patients while a 10-microliters sample volume increased the positives diagnosed to 20.5% (24 of 117).