PCR-based Pool Screening Method for High Throughput Wuchereria Bancrofti Detection
News Apr 24, 2013
Effective diagnostic tools are necessary to monitor and evaluate interruption of Lymphatic Filariasis (LF) transmission. Accurate detection of Wuchereria bancrofti (Wb) microfilaria (mf) is essential to measure the impact of community treatment programmes. PCR-based assays are specific, highly sensitive tools allowing the detection of Wuchereria bancrofti DNA in human blood samples. However, current protocols describing the pool screening approach, use samples of less than 60 µl of blood, which limits the sensitivity of the poolscreen PCR assay. The purpose of this study was to improve the pool-screen PCR protocol to enhance its sensitivity and usefulness for population scale studies.
DNA extractions were performed with the DNeasy kit, the PCR with the Wb LDR primers and the SYBR-Green dye. Improvements of our pool-screen real-time PCR (qPCR) assay allowed the detection of as little as one Wb microfilaria diluted in a pool of at least 12 blood samples of 60 µl each. Using this assay, mf burdens can be predicted using a standard curve derived from mf spiked dried blood samples. The sensitivity achieved is equivalent to the detection of a single LF positive individual carrying a mf burden as low as 18 mf/ml, in a pool of blood samples from at least 12 individuals.
Due to its sensitivity, rapidity and cost-effectiveness, we suggest this qPCR pool-screening assay could be used as a diagnostic tool for population- scale filariasis elimination monitoring and evaluation.
This article is published online in the Journal Parasites and Vectors and is free to access.