Sensitive Molecular Blood Test Detects Malaria

A real time polymerase chain reaction (PCR) assay for Plasmodium has been designed to overcome the problems of inhibition in molecular testing.

Real-time PCR is a sensitive and specific method for the analysis of Plasmodium DNA. However, prior purification of genomic DNA from blood is necessary since PCR inhibitors and quenching of fluorophores from blood prevent efficient amplification and detection of PCR products.

Scientists from the University of Alberta (Edmonton, AB, Canada) obtained 67 blood samples from febrile patients with suspected malaria between 2008 and 2011 following diagnosis by microscopy and routine testing by real-time PCR. Dried blood spots were prepared on filter paper from blood samples collected from patients who attended a malaria clinic in Colombia between 2008 and 2010.

Blood was added directly to the PCR reaction mix and amplified by real-time PCR, which was performed on the ABI 7500 Fast system (Applied Biosystems; Carlsbad, CA, USA). Spots excised from filter paper were either directly added to the PCR master mix or washed briefly in water. Amplification of Plasmodium DNA was detected in both the neat and washed filter papers, although the amplification curves for the neat samples had lower background and tighter replicates compared with the washed samples.

Amplification and fluorescence detection by real-time PCR were optimal with 40× SYBR Green dye (Invitrogen Life Technologies; Carlsbad, CA, USA), and 5% blood volume in the PCR reaction. Malarial DNA was detected directly from both whole blood and dried blood spots from clinical samples. The sensitivity and specificity ranged from 93% to 100% compared with PCR performed on purified Plasmodium DNA and all four species of malaria could be detected.

The authors of the study concluded that the methodology they described facilitates high-throughput testing of blood samples collected in the field by fluorescence-based real-time PCR. This method can be applied to a broad range of clinical studies with the advantages of immediate sample testing, lower experimental costs, and timesavings. The study was published online on August 19, 2011, in the Malaria Journal.

Related Links:
University of Alberta
Applied Biosystems
Invitrogen Life Technologies