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

Abstract

A quantitative, competitive polymerase chain reaction (QC-PCR) assay for the sensitive detection of Wuchereria bancrofti DNA was developed. A competitor sequence was constructed by an exchange of nucleotides in the Wuchereria-specific Ssp I repeat. The PCR products were hybridized to specific DNA probes and their amounts, determined by an enzyme-linked immunosorbent assay (ELISA). 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 200 microl of blood lysate. The assay was also tested on 78 blood samples collected 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 addition, one mf-negative but antigen-positive sample was also positive as determined by QC-PCR-ELISA. A positive correlation of mf density with the QC-PCR-ELISA 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 of 84.6 units. Because of the high standard deviation within each group, estimates of worm burdens in infected individuals using the QC-PCR-ELISA are not recommended. However, we present data indicating that the W. bancrofti QC-PCR-ELISA is a powerful new tool for evaluation of parasitic loads for community-based diagnosis of bancroftian filariasis.

Evaluation of PCR-ELISA as a tool for monitoring transmission of Wuchereria bancrofti in District of Gampaha, Sri Lanka

“…quinquefasciatus mosquitoes were collected in [15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30] households in each study site once a year. Initially, an index household was selected based on the presence of a microfilaria carrier (already treated for microfilaria) among the household residents within the past 5 years.…”

“…Female Cx. quinquefasciatus mosquitoes were divided into batches of [15][16][17][18][19][20] mosquitoes for the analysis by PCR and dissection. Mosquito batches for dissection were analyzed immediately and the rest were desiccated and stored at -20 °C until further analysis.…”

“…PCR products were detected by ELISA as described by Fischer and colleagues with minor modifications [18] . The PCR product ( 405 nm following a one-hour incubation at 37 °C using a Vmax microplate reader (Molecular Devices, Sunnyvale, CA).…”

Application of a polymerase chain reaction-ELISA to detect Wuchereria bancrofti in pools of wild-caught Anopheles punctulatus in a filariasis control area in Papua New Guinea.

“…Each amplification reaction was performed in a final volume of 50 l containing two oligonucleotide primers, NV-1, NV-2, and 100 fg of an internal plasmid-based control. 16 The sequences of these primers were NV-1: 5Ј-CGTGATGGCATCAAAGTAGCG-3Ј (21-mer) and NV-2: 5Ј-CCCTCACTTACCATAAGA-CAAC-3Ј (22-mer). The NV-2 primer (reverse) used was biotinylated at the 5Ј end to obtain a PCR product that can bind to a streptavidin-coated microtiter plate.…”

“…This procedure was performed according to the detailed protocol developed by Fischer and others. 16 Microtiter plates (Lab-Systems, Needham Heights, MA) were coated overnight at 4ЊC using 1 g/ml of streptavidin (Sigma, St. Louis, MO) in coating buffer (0.1 M Na 2 C0 3 -NaHCO 3 , pH 9.6). For duplicate testing of each sample (once with control hybridization probe and once with the wild-type Ssp I-specific hybridization probe), 40 l of the biotinylated PCR product from each pool was mixed with 360 l of hybridization buffer (6ϫ SSPE [0.8 M sodium phosphate, pH 8.3, 5 mM EDTA]), 5ϫ Denhardt's solution (0.01% ficoll, 0.01% polyvinylpyrrolidone, 0.01% bovine serum albumin), 0.1% sodium sarcosine, 0.02% sodium dodecyl sulfate, 0.05% NaN 3 ).…”