Real-Time Fluorescence-Based PCR for Detection of Malaria Parasites

Abstract

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...

Development of a Real-Time PCR Assay for Detection of Plasmodium falciparum , Plasmodium vivax , and Plasmodium ovale for Routine Clinical Diagnosis

“…in blood samples (4,8). The real-time PCR developed in this study allows the simultaneous detection and quantification of P. falciparum, P. vivax, and P. ovale, the three parasite species responsible for most malaria admissions in areas of endemicity as well as areas of nonen- a Malaria species are classified according to the diagnosis obtained from the blood slide examination.…”

“…Real-time PCR-a new methodology that employs fluorescent labels to enable the continuous monitoring of amplicon (PCR product) formation throughout the reaction-has recently been adapted to detect all four human malaria parasites indiscriminately and screen large numbers of samples (8), while another real-time PCR-based method allows quantitative detection of P. falciparum DNA in blood samples (4).…”

Ebola viral load at diagnosis associates with patient outcome and outbreak evolution

“…The assays detected EBOV at approximately 10 genome equivalents/reaction. Differential diagnostics were also offered for Lassa virus (18) and Plasmodium species (19). As an internal control for extraction and amplification procedures, MS2 phage was added to each sample as previously described (20) to ensure that the extraction, PCR setup, and run parameters did not change between operators or over time.…”

Use of Molecular Beacon Probes for Real-Time PCR Detection of Plasmodium falciparum and Other Plasmodium Species in Peripheral Blood Specimens

“…With a total assay time of 2 h, our assays are sufficiently robust for routine clinical use. To date, no other molecular beacon probe-based real-time PCR assays for malaria diagnosis have been reported prior to ours, although other investigators have described their experience with TaqMan probes (9,10,17,18) and SYBR green dye (4,6,12). The Plasmodium 18S (small-subunit) rRNA gene has served as the prototypic target for genus-based malaria PCR assays (2-4, 6, 7, 9-12, 14, 16, 18), although the cox1 gene has not previously been used for P. falciparum malaria identification.…”