A pond-side test for Guinea worm: Development of a loop-mediated isothermal amplification (LAMP) assay for detection of Dracunculus medinensis.

Guinea worm Dracunculus medinensis causes debilitating disease in people and is subject to an ongoing global eradication programme. Research and controls are constrained by a lack of diagnostic tools. We developed a specific and sensitive LAMP method for detecting D. medinensis larval DNA in copepod vectors. We were able to detect a single larva in a background of field-collected copepods. This method could form the basis of a "pond-side test" for detecting potential sources of Guinea worm infection in the environment, in copepods, including in the guts of fish as potential transport hosts, enabling research, surveillance and targeting of control measures. The key constraint on the utility of this assay as a field diagnostic, is a lack of knowledge of variation in the temporal and spatial distribution of D. medinensis larvae in copepods in water bodies in the affected areas and how best to sample copepods to obtain a reliable diagnostic sample. These fundamental knowledge gaps could readily be addressed with field collections of samples across areas experiencing a range of worm infection frequencies, coupled with field and laboratory analyses using LAMP and PCR.

The development of monoclonal antibodies to the secA protein of Cape St. Paul wilt disease phytoplasma and their evaluation as a diagnostic tool.

Partial recombinant secA proteins were produced from six different phytoplasma isolates representing five 16Sr groups and the expressed, purified recombinant (partial secA) protein from Cape St. Paul wilt disease phytoplasma (CSPWD, 16SrXXII) was used to immunise mice. Monoclonal antibodies (mAbs) were selected by screening hybridoma supernatants for binding to the recombinant proteins. To characterise the binding to proteins from different phytoplasmas, the antibodies were screened by ELISA and western blotting, and epitope mapping was undertaken. Eight different mAbs with varying degrees of specificity against recombinant proteins from different phytoplasma groups were selected. Western blotting revealed that the mAbs bind to proteins in infected plant material, two of which were specific for phytoplasmas. ELISA testing of infected material, however, gave negative results suggesting that either secA was not expressed at sufficiently high levels, or conformational changes of the reagents adversely affected detection. This work has shown that the phytoplasma secA gene is not a suitable antibody target for routine detection, but has illustrated proof of principle for the methodology.