Fig. 4.

UPDx has a limit of detection similar to standard qPCR. a Serial dilutions of P. falciparum 3D7 parasites in whole human blood were processed using the original universal parasite detection method (grey bars), the nested method with DNA Digest 1 only (light-colored bars), the nested method with DNA Digest 2 only (medium-colored bars), or the nested method with both DNA Digest 1 and DNA Digest 2 (dark bars). Statistical significance of differences between conditions was assessed using a 2-way ANOVA with Tukey’s multiple comparisons posttest, p < 0.0001, n = 3, mean ± SD. b Nested PCR with both restriction Digest 1 and Digest 2 detected P. falciparum at a limit of 0.58 parasites/μL in 2/3 samples (each point is the average of 2 or 3 replicates, error bars are ± 1 SD, circles = parasites were detected in 3/3 replicates, squares = parasites were detected in 2/3 replicates, and triangles = parasites were detected in fewer than 2 replicates). c Analysis of the same samples by qPCR demonstrated that positive amplification curves were observed between 0.58 and 5.8 parasites/μL (each point represents the average of three replicates, error bars are ± SD, diamonds are positive controls; Pos. 1 and Pos. 2, circles = detection of parasites in 3/3 replicates, squares = detection of parasites in 2/3 replicates, and triangles = detection of parasites in 0/3 replicates). Percentage total reads refers to the percentage of all reads generated that were used to construct haplotypes identical to P. falciparum 18S sequences with the GenBank Accessions XR_002966654.1 and XR_002273081.2