Detection and Differentiation of Filarial Parasites by Universal Primers and Polymerase Chain Reaction–restriction Fragment Length Polymorphism Analysis

Abstract

Filarial nematode parasites are a serious cause of morbidity in humans and animals. Identification of filarial infection using traditional morphologic criteria can be difficult and lead to misdiagnosis. We report on a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP)-based method to detect and differentiate a broad range of filarial species in a single PCR. The first internal transcribed spacer 1 (ITS1) along with the flanking 18S and 5.8S ribosomal DNA (rDNA) were isolated and cloned from Wuchereria bancrofti, Brugia malayi, and Brugia pahangi. Sequence analysis identified conserved sites in the 18S and 5.8S rDNA sequence that could be used as universal priming sites to generate ITS1-distinctive PCR products that were useful for distinguishing filariae at the genus level. The addition of a digestion of the ITS1 PCR product with the restriction endonuclease Ase I generated a fragment profile that allowed differentiation down to the species level for W. bancrofti, B. malayi, B. pahangi, Dirofilaria immitis, and D. repens. The PCR-RFLP of ITS1 rDNA will be useful in diagnosing and differentiating filarial parasites in human, animal reservoir hosts, and mosquito vectors in disease-endemic areas.

Nested PCR to detect and distinguish the sympatric filarial species Onchocerca volvulus, Mansonella ozzardi and Mansonella perstans in the Amazon Region

“…Unlike the PCR-RFLP method developed by Nuchprayoon et al (2005), our assay does not digest the PCR product with restriction endonucleases and increases sensitivity using a nested amplification.…”

“…Only a small subset of methods is able to differentiate several nematodes in the same PCR reaction, generally after enzymatic digestion (Nuchprayoon et al 2005).…”

Detection of Onchocerca volvulus in Skin Snips by Microscopy and Real-Time Polymerase Chain Reaction: Implications for Monitoring and Evaluation Activities

“…methods include the O-150 PCR-ELISA used here, qPCR-MCA of the O-150 repeat, TaqMan-based qPCR, loopmediated isothermal amplification, specific oligonucleotide capture with magnetic beads, nested PCR combining general and species-specific primers, and PCR with general filarial primers followed by restriction fragment length polymorphism analysis to determine taxonomic identity. 13,[15][16][17][18][19][20][21][22][23] However, with few exceptions, these procedures require at least one post-amplification step for final determination, are specific to only one genus, or require multiple specific reaction components. 16,17 The procedure used here allows for singletube assessment of both presence and identity in a single 90-minute reaction.…”

A Novel Dirofilaria Species Causing Human and Canine Infections in Hong Kong

“…Therefore, since our isolates have a 4% nucleotide difference from the reported D. repens sequence and an 11% difference from the D. immitis sequence, they belong to a separate species. The 18S-ITS1-5.8S gene cluster has also been employed to differentiate between filariae at the genus level (18,19). A novel species, Acanthocheilonema ladakhii, was described based on the 18S ribosomal DNA (rDNA) gene sequence (15).…”