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

Abstract

We present filaria-nested polymerase chain reaction (PCR)

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.…”

“…Conserved tracts within the ITS1 and 5.8S regions of the rDNA gene and flanking a polymorphic segment of the ITS1were targeted for primer design (ITS1-UNIF: 5′-GGTGwTATTCGTTGG TGTCTAT-3′ and 5.8S-EXTR: 5′-AGCTAGCTGCGTTCTT CAT-3′, the latter primer being a modified version of the UNI-1R primer), resulting in a 246-270 bp amplicon corresponding to the 3′ end of the ITS1. 13 Known larval and/ or adult isolates of Dirofilaria immitis, Brugia malayi, and O. volvulus were used to characterize specific melt curve profiles of the amplicon and establish the ability of the assay to differentiate species on the basis of melt temperature (T m ). Although neither D. immitis nor B. malayi are expected to have epidemiologic relevance to human onchocerciasis, availability of well-characterized biological and genetic materials through the Filariasis Research Reagent Resource Center makes these species well-suited for inclusion as technical controls for a pan-filarial assay.…”

“…15 Finally, for quality control of qPCR-MCA specificity, a randomly selected subset of 24 qPCR-MCA-positive skin snip samples were also subjected to standard Sanger sequencing of the entire ITS1 (external primers 18S-EXTF: 5′-TGCTGTAACCATTACCGAAAG-3′ modified from and 5.8S-EXTR as above; internal primers 18S-INTF: 5′-TGAG CCGTTTCGAGAAAAGC-3′, ITS1-UNIR: 5′-TyAGGCG ATAAyTTGGATAAAG-3′, and ITS1-UNIF as above). 13 In brief, externally amplified PCR product was purified (StrataPrep PCR Purification kit; Agilent Technologies), sequenced bidirectionally with internal primers and BigDye ® Terminator v3.1 chemistry (LifeTechnologies, Inc.), and analyzed on an ABI 3130xl genetic analyzer (LifeTechnologies, Inc.). Electropherograms were evaluated and trimmed manually and assembled in ChromasPro v1.7.6 (Technelysium, South Brisbane, Australia) before interrogation of the National Center for Biotechnology Information's nucleotide database via the Basic Local Alignment Search Tool using the megablast algorithm.…”

“…As previously described, length polymorphisms of the first internal transcribed spacer (ITS1) of the ribosomal DNA (rDNA) repeat can be used for the detection and differentiation of filarial parasite species. [11][12][13] Highly conserved regions within the ITS1 and in the flanking 18S and 5.8S tracts allow for development of "generalist" primers that can be used across related species. Thus, with a single set of primers, DNA from an array of filarial species can be amplified and then differentiated on the basis of amplicon length or sequence polymorphism.…”

Filarial Chyluria as a Rare Cause of Urinary Retention

“…A molecular analysis of the pathogenic parasite was performed according to a previously reported method (8). A QIAamp DNA mini kit (QIAGEN, Hilden, Germany) was used for DNA extraction from whole blood samples.…”