A fluorescence-based real-time 5 nuclease PCR capable of detecting all four human malaria parasites was developed to screen large numbers of samples during an outbreak to prevent further transmission of malaria. The effectiveness of antimalarial therapy for malaria patients can be monitored by determining the reduction of parasitemia by this method.Malaria is a major tropical infection causing up to 500 million clinical cases and 2.7 million deaths per year worldwide. For proper treatment of malaria patients, accurate and rapid diagnosis of malaria is essential. The microscopic examination of a blood smear is the "gold standard" for malaria diagnosis. The method is sensitive and specific but laborious and timeconsuming (6). Alternative methods, such as PCR and rapid antigen capture assays (ParaSight F test, OptiMAL rapid malaria test, and ICT Malaria P.f/P.v), have been developed. A PCR to detect Plasmodium falciparum and P. vivax was previously developed and evaluated (1,7,8,9). We have developed a method for malaria diagnosis that can be adapted for highthroughput rapid screening of hundreds of samples with a sensitivity and specificity comparable to those of the microscopic method. The method is a fluorescence-based real-time 5Ј nuclease PCR based on the TaqMan technology (Roche Molecular Diagnostics Systems) (3, 4), with primers and a probe from the small-subunit (SSU) rRNA gene.Malaria samples. Clinical blood samples were obtained from the Malaria Reference Center in the Department of Microbiology, National University of Singapore. Clinical samples of patients undergoing treatment were obtained from the National University Hospital, Singapore. The Gombak A strain of P. falciparum (5) was cultured by the method of Trager and Jensen (10). Two P. ovale (Po1 and Po2) and three P. malariae (Pm1, Pm2, and Pm3) samples were obtained.DNA extraction. DNA was extracted from 200 l of EDTAtreated blood samples with a QIAamp DNA blood minikit (Qiagen GmbH, Hilden, Germany) and stored at 4°C.Sequences of primers and probes. Primers (Mach 60 [5Ј-A CATGGCTATGACGGGTAACG-3Ј] and Mach 61 [5Ј-TGC CTTCCTTAGATGTGGTAGCTA-3Ј]) and a probe (Mach 62 [5Ј-TCAGGCTCCCTCTCCGGAATCGA-3Ј]) were designed to detect the SSU rRNA genes of all four human malarial species (accession numbers M19172 for P. falciparum, X13926 for P. vivax, M54897 for P. malariae, and L48987 for P. ovale). The probe was labeled with a reporter dye, FAM (5-carboxyfluorescein), and a quencher dye, TAMRA (N,N,N,N-tetramethyl-6-carboxyrhodamine) (Applied Biosystems, Foster City, Calif.). 5 nuclease PCR. Five microliters of DNA, 900 nM Mach 60, 300 nM Mach 61, 200 nM Mach 62, and 1ϫ TaqMan universal PCR master mix (Applied Biosystems) in a total reaction volume of 25 l were amplified on an iCycler (Bio-Rad Laboratories, Hercules, Calif.). The following conditions were used for PCR: an initial denaturation at 50°C for 2 min, 95°C for 10 min, and 45 cycles of 95°C for 15 s and 60°C for 1 min. The cycle threshold value (C T ) indicates the cycle where the fluorescence detect...

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