RESUMEN
The internal transcribed spacer (ITS) region was used to study the intraspecies variation of Brugia spp. in cat reservoirs. Blood specimens from seven naturally infected cats were collected from two different geographical brugian-endemic areas in Thailand. The DNAPAR tree of these Brugia spp. was constructed using a maximum likelihood approach based on ITS nucleotide sequences and was compared to those of Brugia malayi, Brugia pahangi, and Dirofilaria immitis that were previously reported in GenBank. The phylogenetic trees inferred from ITS1, ITS2, and complete ITS sequences indicated that B. malayi and B. pahangi were separated into two clades, and subgroups were generated within each clade. The data revealed that ITS2 sequences were less informative than ITS1 for studying intraspecies variation of Brugia spp. Our results are primary data for intraspecies variation of B. malayi and B. pahangi in cat reservoirs. The information could be applicable for studying the molecular epidemiology and the dynamic nature of the parasites.
Asunto(s)
Biodiversidad , Brugia Malayi/clasificación , Brugia pahangi/clasificación , Enfermedades de los Gatos/parasitología , ADN Espaciador Ribosómico/genética , Reservorios de Enfermedades , Filariasis Linfática/veterinaria , Animales , Sangre/parasitología , Brugia Malayi/genética , Brugia Malayi/aislamiento & purificación , Brugia pahangi/genética , Brugia pahangi/aislamiento & purificación , Gatos , ADN de Helmintos/química , ADN de Helmintos/genética , ADN Espaciador Ribosómico/química , Filariasis Linfática/parasitología , Datos de Secuencia Molecular , Filogenia , Análisis de Secuencia de ADN , TailandiaRESUMEN
OBJECTIVE: To identify two closely related Brugia malayi and B. pahangi in cat reservoirs by using high resolution melting real-time PCR (HRM real-time PCR). MATERIAL AND METHOD: HRM analysis on the Corbett Rotor-Gene 6000 instrument was used to test 5 Brugia specimens by using five sets of specific primers for HhaI repetitive region (HR), small heat shock protein (SHP), small subunit ribosomal DNA (18S rDNA), internal transcribed spacer region (ITS), and trans-spliced leading Exon I gene (SLX1). RESULTS: HRM analysis of ITS and SLX clearly generated 2 profiles of B. malayi and B. pahangi while those of HR, 18S rDNA, and SHP could classify B. pahangi. CONCLUSION: HRM is a simple and rapid method for identification of two closely related B. malayi and B. pahangi in which it can detect both parasites within 30 min after real-time PCR detection. This assay is probe-free HRM and reduces a risk of PCR carryover. It does not require multiplex methods and DNA sequencing; therefore, HRM provides a new approach for genetic screening and rapid detection of closely related species in a clinical laboratory.