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PCR-based Pool Screening Method for High Throughput Wuchereria Bancrofti Detection

Published: Wednesday, April 24, 2013
Last Updated: Wednesday, April 24, 2013
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The purpose of this study was to improve the DNA extraction protocol and qPCR assay to achieve the detection of a single mf in a pool of at least twelve 60-µl blood samples.

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.

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