Optimization of Copepod DNA Extraction in Support of Programmatic Surveillance for Guinea Worm Disease

Abstract

The number of cases of guinea worm disease have declined from the millions to the tens over the past few decades, thanks to the efforts of the Carter Center. Their goal, however, is eradication of the disease, which they have remained just shy of for many years. To close these final remaining gaps, environmental surveillance-inspired tactics may be useful to understand disruption of transmission and prevent cases before they happen. As part of this surveillance, the detection of cyclopoid copepods, guinea worm’s intermediate host, would allow for increased sample validity control. There is additional need for species-specific data of the copepods to further inform the ecology of transmission. To this end, a qPCR assay to detect cyclopoid copepods in a body of water would help inform prevention strategies. As a first step toward assay development, DNA extraction methods from copepods must be established and validated. In this study, nine DNA extraction methods were tested and compared. A pre-treatment step was also developed to aid in copepod lysis, which used a combination of enzymatic (Proteinase K and chitinase) and mechanical (homogenization using a micropestle) methods. NEB’s Monarch Spin gDNA kit produced the highest mean DNA yield in the comparison experiment (6.20 ng/µL), followed by a magnetic bead purification method (4.01 ng/µL), and a NaOH heat shock method (1.82 ng/µL). Based on these yields, as well as comparisons of costs and requirements of resources, time, hazardous materials, and skill, the methods were down selected to the NaOH heat shock method and the Monarch Spin gDNA kit.