A real-time PCR-based assay for detection of Wuchereria bancrofti DNA in blood and mosquitoes

Abstract

We developed and evaluated real-time polymerase chain reaction (PCR) assays for detecting Wuchereria bancrofti DNA in human blood and in mosquitoes. An assay based on detection of the W. bancrofti "LDR" repeat DNA sequence was more sensitive than an assay for Wolbachia 16S rDNA. The LDR-based assay was sensitive for detecting microfilarial DNA on dried membrane filters or on filter paper. We also compared real-time PCR with conventional PCR (C-PCR) for detecting W. bancrofti DNA in mosquito samples collected in endemic areas in Egypt and Papua New Guinea. Although the two methods had comparable sensitivity for detecting filarial DNA in reference samples, real-time PCR was more sensitive than C-PCR in practice with field samples. Other advantages of real-time PCR include its high-throughput capacity and decreased risk of cross-contamination between test samples. We believe that real-time PCR has great potential as a tool for monitoring progress in large-scale filariasis elimination programs.

Figure 1

This figure shows the 195 bp SspI repeat sequence of W. bancrofti within the LDR sequence and the species-specific sequences of primers selected for amplification of SspI (NV1, NV2) by C-PCR and LDR (LDR1, LDR2, TaqMan probe) by real-time PCR. Oligonucleotide sequences are shown in the 5′ to 3′ orientation.

Figure 2

Amplification plots of fluorescence (y-axis) vs cycle number (x-axis) show the analytical sensitivity of the LDR real-time PCR for detecting W. bancrofti DNA. Genomic DNA template from W. bancrofti microfilariae (MF) isolated from membrane filters (10, 1, 0.1, 0.01, 0.001 ng) were tested in duplicate. Cycle threshold values (Ct) (y-axis) were plotted against Log template DNA (x-axis) concentrations (range, 1 pg to 10 ng) to generate a standard curve with reproducible linearity over five orders of magnitude (inset); 1 pg is approximately equivalent to 0.5% of the DNA found in a single W. bancrofti MF (S. Williams, unpublished data).

Figure 3

This graph compares the sensitivity of real-time PCR for detection of W. bancrofti DNA in blood for two target sequences: W. bancrofti LDR and Wolbachia 16S rDNA. Genomic DNA samples were isolated from nucleopore membrane filters containing microfilariae (MF) filtered from night blood samples. Ct values are plotted against Log MF counts (MF/ml). Samples that failed to reach the fluorescence threshold by 40 cycles were considered to be negative, and these were not plotted in the graph. The LDR assay was positive in 19 of 19 samples (100%); Ct values were significantly correlated with MF numbers (R = −0.560, P = 0.013). The 16S rDNA assay detected the target in 11 of 19 samples (56%; correlation between Ct and MF counts: R = −0.436, P = 0.180, not significant).

Figure 4

This graph compares the percentage of mosquito pools with W. bancrofti DNA detected by real-time PCR in two sentinel Egyptian villages Tahoria (TH) and Kafr El Bahary (KB) before and after three rounds of MDA with antifilarial medication. Microfilaria prevalence rates as determined by membrane filtration in these villages at these time-points were 4.2% and 0% for TH and 10.4% and 1.4% for KB, respectively. The number of mosquito pools tested before and after MDA were 121 and 100 from TH and 105 and 93 from KB, respectively. No positive mosquito pools were detected in TH after the third round of MDA.