Figure 1.
18S ribosomal RNA gene based P. knowlesi primer cross-reacts with P. vivax and other simian-infecting malaria species.
Published 18S ribosomal RNA gene based P. knowlesi primers [3] were used to test 5 different simian-infecting malaria parasite species (P. simiovale, P. inui, P. cynomolgi, P. hylobati and P. coatneyi) including 3 different P. knowlesi isolates (A) and 11 P. vivax strains (B). A no template control (NTC) was included. Cross reactivity was observed with some of the simian malaria parasites and some P. vivax strains.
Table 1.
Sequence of the novel Pkr140-5 primer set.
Figure 2.
Primer Pkr140-5 tested with the 4 human-infecting malaria species.
To test the specificity of the novel primers, DNA from the four additional human-infecting Plasmodium parasites were tested. P. knowlesi (H strain) was used as a positive control (expected size = 200bp). A no template control (NTC) was also included.
Figure 3.
Specificity of the P. knowlesi primers tested using simian-infecting malaria species.
To test the specificity of the P. knowlesi primers, 5 different simian-infecting malaria parasite species (P. simiovale, P. inui, P. cynomolgi, P. hylobati and P. coatneyi) including 3 different P. knowlesi isolates were tested. The no template control (NTC) was included as a negative control. A; primer set Pkr140-3 (expected size = 230bp), B; primer set Pkr140-4 (expected size = 280bp) and C; primer set Pkr140-5 (expected size = 200bp). Circles indicate non-specific amplification.
Figure 4.
Primer set Pkr140-5 does not cross react with P. vivax.
Multiple P. vivax strains were tested using primer set Pkr140-5 (expected size 200bp) in order to test the primers' specificity. NTC = No template control.
Figure 5.
Limits of detection of primer set Pkr140-5.
The analytical sensitivity of primer set Pk140-5 (A) and the primers from a published study [3] (B) were determined using a well-quantitated P. knowlesi DNA standard. The blood sample was serially diluted ten-fold with a starting parasitemia of 100,000p/µl to 1p/µl. The expected base pair sizes for the two primers are included. Three different experiments are shown.
Figure 6.
Spatial distribution of Pkr140 sequence targets across the 14 P. knowlesi genome.
The circle represents chromosomes. Each chromosome is labeled with the 2-letter genus and species abbreviation for P. knowlesi and the chromosome number. Tick marks indicate 1 mb of sequence. Lines inside the circle indicate the location of Pkr140 copies and are not to scale. Circos 0.51 (http://mkweb.bcgsc.ca/circos/) was used to generate this map [44].