Rapid detection and identification of Wuchereria bancrofti, Brugia malayi, B. pahangi, and Dirofilaria immitis in mosquito vectors and blood samples by high resolution melting real-time PCR

Abstract

A simple, rapid, and high-throughput method for detection and identification of Wuchereria bancrofti, Brugia malayi, Brugia pahangi, and Dirofilaria immitis in mosquito vectors and blood samples was developed using a real-time PCR combined with high-resolution melting (HRM) analysis. Amplicons of the 4 filarial species were generated from 5S rRNA and spliced leader sequences by the real-time PCR and their melting temperatures were determined by the HRM method. Melting of amplicons from W. bancrofti, B. malayi, D. immitis, and B. pahangi peaked at 81.5±0.2℃, 79.0±0.3℃, 76.8±0.1℃, and 79.9±0.1℃, respectively. This assay is relatively cheap since it does not require synthesis of hybridization probes. Its sensitivity and specificity were 100%. It is a rapid and technically simple approach, and an important tool for population surveys as well as molecular xenomonitoring of parasites in vectors.

Keywords: Brugia malayi; Brugia pahangi; Dirofilaria immitis; Wuchereria bancrofti; dog; high resolution melting real-time PCR; mosquito.

Fig. 1

Amplification plot of fluorescence (y-axis) vs cycle numbers (x-axis) showing the analytical sensitivity of HRM real-time PCR for detecting DNA of W. bancrofti (A), B. malayi (B), D. immitis (C), and B. pahangi (D) in control plasmids. (a-h) 10-fold dilutions of W. bancrofti plasmid from 9×10⁷ to 9 copies/reaction (i) distilled water. (j-q) 10-fold dilutions of B.malayi plasmid from 2×10⁷ to 2 copies/reaction (r) distilled water. (s-y) 10-fold dilutions of D. immitis plasmid from 10⁷ to 10 copies/reaction (z) D. immitis plasmid 1 copy/reaction and distilled water. (I-VIII) 10-fold dilutions of B. pahangi plasmid from 4×10⁷ to 4 copies/reaction (IX) distilled water.

Fig. 2

Representative melting curve analyses of the amplification products of W. bancrofti, B. malayi, D. immitis, and B. pahangi 5S rRNA gene and spliced leader sequence. The figure shows the melting curves of D. immitis plasmid DNA (10⁶ copies) (a), D. immitis-infected blood sample (b), B. malayi plasmid DNA (2×10⁴ copies) (c), B. malayi-infected blood sample (d), B. pahangi plasmid (4×10⁴ copies) (e), B. pahangi-infected blood sample (f), W. bancrofti plasmid (9×10⁴ copies) (g), W. bancrofti-infected blood sample (h), non-infected Ae. aegypti and Cx. quinquefasciatus, P. falciparum-infected and P. vivax-infected human red blood cells, non-infected cat, human, and dog blood samples, Babesia spp., and H. canis-infected dog blood DNA, respectively, and distilled water (all i).