Validation of an apicoplast genome target for the detection of XXXX species using polymerase chain reaction and loop mediated isothermal amplification.

@entity311 @entity30261 @entity1098
UNASSIGNED: The genome of the @entity30261 apicoplast, which has a higher copy number compared with current targets for molecular diagnosis of @entity311 , appears to be a suitable target for detection of submicroscopic infections that are capable of sustaining transmission. Novel primers targeting a conserved segment of the apicoplast (PFC10_AP 0010:rRNA) were designed and used in a number of different high throughput platforms such as single-step PCR (ssPCR), nested PCR (nPCR) and loop-mediated isothermal amplification (LAMP) for parasite detection. Replicates of ten-fold serial dilutions of @entity30261 DNA, with equivalent parasite density ranges of 200 000 to 0.2 parasites/ L, were used to determine the limit of detection and repeatability of each assay. A panel of 184 archived DNA samples extracted from either @entity1098 whole blood or dried blood spots, from across West Africa and South East Asia was used to determine the diagnostic performance of the assays. All assays amplified the 2 parasites/ L dilution except the ssPCR, which amplified two of the three replicates. Using an 18S rRNA PCR as reference, the sensitivity was 98% (95% CI 93-100%) for the LAMP assay, 87% (95% CI 79-93%) for ssPCR and 100% (95% CI 97-100%) for nPCR. Specificity was 91% (95% CI 83-96%) for LAMP, 82% (95% CI 72-90%) for ssPCR and 66% (95% CI 54-76%) for nPCR. The apicoplast genome-based nPCR detected more positive samples overall than the reference method. Discrepant samples were confirmed as true positives using a probe-based real-time quantitative PCR assay. The results show that the apicoplast genome is a suitable target for molecular diagnosis of @entity311 .
