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+/-0.2degrees C, 79.0+/-0.3degrees C, 76.8+/-0.1degrees C, and 79.9+/-0.1degrees C, 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.
Subject(s)
Animals , Cats , Dogs , Humans , Male , Blood/parasitology , Brugia/classification , Culicidae/parasitology , Dirofilaria immitis/classification , Parasitology/methods , RNA, Helminth/genetics , RNA, Ribosomal, 5S/genetics , Real-Time Polymerase Chain Reaction/methods , Sensitivity and Specificity , Transition Temperature , Wuchereria bancrofti/classificationABSTRACT
The first human case with trichinellosis was reported in 1964 in Tibet, China. However, up to the present, the etiological agent of trichinellosis has been unclear. The aim of this study was to identify a Tibet Trichinella isolate at a species level by PCR-based methods. Multiplex PCR revealed amplicon of the expected size (173 bp) for Trichinella spiralis in assays containing larval DNA from Tibet Trichinella isolate from a naturally infected pig. The Tibet Trichinella isolate was also identified by PCR amplification of the 5S ribosomal DNA intergenic spacer region (5S ISR) and mitochondrial large-subunit ribosomal RNA (mt-lsrDNA) gene sequences. The results showed that 2 DNA fragments (749 bp and 445 bp) of the Tibet Trichinella isolate were identical to that of the reference isolates of T. spiralis. The Tibet Trichinella isolate might be classifiable to T. spiralis. This is the first report on T. spiralis in southwestern China.
Subject(s)
Animals , Humans , DNA, Helminth/chemistry , DNA, Mitochondrial/chemistry , DNA, Ribosomal/chemistry , DNA, Ribosomal Spacer/genetics , Genotype , Multiplex Polymerase Chain Reaction , RNA, Ribosomal, 5S/genetics , Sequence Analysis, DNA , Swine , Swine Diseases/parasitology , Tibet , Trichinella spiralis/classification , Trichinellosis/parasitologyABSTRACT
Our research work group has been interested in the study of the ribosomal RNA and 5S gene systems from Trypanosoma cruzi. Our contributions span from the general description of a multifragmented molecular system, to the sequence analysis of some ribosomal RNA coding regions. From the latter, we have constructed phylogenetic trees of the Trypanosomatidae family, and our data indicate that the molecular inferences do not sustain the traditional classification of these species. Our published findings are here reviewed along with recent unpublished observations of ribosomal RNA and 5S gene structures