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Fig. 1 | Infectious Diseases of Poverty

Fig. 1

From: A direct, sensitive and high-throughput genus and species-specific molecular assay for large-scale malaria screening

Fig. 1

Workflow of multi-section CLIP-PCR assay (mCLIP-PCR) for Plasmodium genus and species screening. Dried blood samples are first pooled and lysed. Depicted here is a 10 × 10 matrix-pooling, where samples in the same row are pooled and tested together as one, and so are the samples in the same column. In this way all 100 samples are analyzed twice just by testing Pool A to J and Pool 1–10 (in a total of 20 tests). The released target RNAs from both P. falciparum (Pf) and P. vivax (Pv) are captured to the same well in a 96-well plate via hybridization with oligo probes on multiple sections of each target RNA. Sections include either conserved (“Genus”) or species-specific (“Pf” or “Pv”) sequences of each target RNA. Within each section, probes bind contiguously to the target. Capture Probes (which includes a target-specific sequence and a common tail sequence) at the two ends of a section anchors the target to solid surface by hybridizing the tail sequence with oligos conjugated on the solid surface. After washing, the Detection Probes between the two Capture Probes are ligated to form a single-stranded DNA in each section with both ends having defined sequences as corresponding PCR primer sites. The ligation products are heat-released from the well bottom and 5 μl of which transferred to a new plate for genus PCR amplification using genus-specific primer set (green) and SYBR green chemistry. For the genus-positive sample, additional 5 μl of released ligation product is separately amplified by P. falciparum- or P. vivax-specific primer set (colored orange and red respectively)

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