Fig. 1.

The schematic overview of pp-IPA. The Pasteur Pipette-assisted isothermal probe amplification (pp-IPA) involves three key steps: lysis and capture, ligation of probes, and amplification of templates (bottom). In the initial lysis and capture step (top left), RNAs were released from the cell after lysis and hybridized to the oligonucleotide hybridization probes (CP and LP) targeting highly conserved region of 18S rRNA of Plasmodium species. Each CP contains a target-specific portion and a universal 3ʹ -tail sequence for binding to the oligos on the inner wall of the Pasteur pipette, while the two LPs, which bind to two contiguous sequences of the target with their target-specific portions, have universal sequences forming stem-loop structure in their respective 5ʹ and 3ʹ portion. After washing off unbound probes, LPs (LP1 and LP2) are ligated to form a complete dumbbell-like template, a process only possible in the presence of captured target RNA. The colorimetric IPA process follows an isothermal amplification using Bst polymerase and one pair of generic primers binding to the predesigned generic loop region of the LP probes (top right). Following IPA, the positive sample reaction mix undergoes a change from pink to yellow, which aids in the visual identification by naked-eye observation, while negative samples maintain a pink coloration (bottom)