The amplification of target parasite DNA in human blood by the polymerase chain reaction

Abstract

Plasmodium falciparum is an intraerythrocytic protozoan which is a major cause of human malaria. The specific and sensitive procedure for Plasmodia species identification is essential for a proper therapeutic purpose and epidemiologic surveillance. Although DNA based detection of this parasite provides high specificity but the failure of its sensitivity make it not promising as a diagnostic tool. In order to enhance the sensitivity of the DNA based detection, the amplification of species specific DNA fragment of P. falciparum via the polymerase chain reaction was investigated. The insert of pBR K1-14, a P. falciparum specific DNA probe which had been constructed in our laboratory, was chosen to be the amplification target. Two oligonucleotide primers were designed from this probe flanking on 206 bp fragment of the target in the parasite genome. By the amplification of this specific 206 bp of P. falciparum DNA sequence by PCR as little as 0.01 pg of the parasite extracted DNA (one half of DNA content from a single parasite) or a single parasite in 20 ul blood was detectable by coupling with DNA hybridization. Optimization of the 206 bp amplification condition, namely 10 ul reaction (0.1 uM each primers, 200 uM each dNTP, 0.2 U Taq polymerase) in 40 cycles comprising of 15 sec at 80 degree C denaturation, 15 sec at 50 degree C annealing and 15 sec at 72 degree C extension, could improve the specificity and the efficiency of the amplification of crude blood sample. By this condition the presence of 20 parasites in 20 ul blood, a level of parasitemia usually undetected by microscopic examination, were detectable by visualization of the ethidium bromide stained band following agarose gel electrophoresis. The sensitivity and specificity of PCR procedure had been investigated by comparing with the results from an expert microscopist. More than 2000 blood samples from several endemic areas of Thailand have been tested. The overall data showed that malaria detection by PCR procedure provided 81% sensitivity, 95.8% specificity with 6.6% disagreement relative to the microscopist result. The disagreement between an expert microscopist compared to another expert microscopist or microscopist at a malaria clinic were 5.75% and 15% respectively.