Development of a quantitative, competitive polymerase chain reaction enzyme linked immunosorbent assay for the detection of Wuchereria bancrofti DNA

A quantitative, competitive polymerase chain reaction (QC-PCR) assay for th e sensitive detection of Wuchereria bancrofti DNA was developed. A competit or sequence was constructed by an exchange of nucleotides in the Wucheria-s pecific Ssp I repeat. The PCR products were hybridized to specific DNA prob es and their amounts, determined by an enzyme-linked immunosorbent assay (E LISA). In laboratory-prepared samples the QC-PCR-ELISA assay was capable of detecting the amount of DNA equivalent to 0.1 microfilaria (mf) added to 2 00 mu l of blood lysate. The assay was also tested on 78 blood samples coll ected in endemic areas in Egypt. All 28 samples that were positive both for mf and for circulating antigen were also QC-PCR-ELISA-positive. In additio n. one mf-negative but antigen-positive sample was also positive as determi ned by QC-PCR-ELISA. A positive correlation of mf density with the QC-PCR-E LISA was observed. Samples containing 10 or fewer mf/ml had a mean relative amount of Ssp I PCR product of 19.7 units, whereas samples with 11-100 mf/ ml had a mean of 36.3 units and those with more than 100 mf/ml had a mean o f 84.6 units. Because of the high standard deviation within each group, est imates of worm burdens in infected individuals using the QC-PCR-ELISA are n ot recommended. However, we present data indicating that the W. bancrofti Q C-PCR-ELISA is a powerful new tool for evaluation of parasitic loads for co mmunity-based diagnosis of bancroftian filariasis.