Capture and Ligation Probe-PCR (CLIP-PCR) for Molecular Screening, with Application to Active Malaria Surveillance for Elimination

摘要

BACKGROUND: Malaria control programs have achieved remarkable success during the past decade. Nonetheless, sensitive and affordable methods for active screening of malaria parasites in low-transmission settings remain urgently needed.METHODS: We developed a molecular screening method, capture and ligation probe-PCR (CLIP-PCR), which achieved the sensitivity of reverse-transcription PCR but eliminated the reliance on RNA purification and reverse transcription. In this method, 18S rRNA of genus Plasmodium is released from blood, captured onto 96-well plates, and quantified by the amount of ligated probes that bind continuously to it. We first used laboratory-prepared samples to test the method across a range of parasite densities and pool sizes, then applied the method to an active screening of 3358 dried blood spot samples collected from 3 low-endemic areas in China.RESULTS: Plasmodium falciparum diluted in whole blood lysate could be detected at a concentration as low as 0.01 parasites/μL, and a pool size of ≤36 did not significantly affect assay performance. When coupled with a matrix pooling strategy, the assay drastically increased throughput to thousands of samples per run while reducing the assay cost to cents per sample. In the active screening, CLIP-PCR identified 14 infections, including 4 asymptomatic ones, with 500 tests, costing US$0.60 for each sample. All positive results were confirmed by standard quantitative PCR.CONCLUSIONS: CLIP-PCR, by use of dried blood spots with a pooling strategy, efficiently offers a highly sensitive and high-throughput approach to detect asymptomatic submicroscopic infections with reduced cost and labor, making it an ideal tool for large-scale malaria surveillance in elimination settings.