Abstract
Triatoma dimidiata is the most important Chagas disease insect vector in Central America as this species is primarily responsible for Trypanosoma cruzi transmission to humans, the protozoan parasite that causes Chagas disease. T. dimidiata sensu lato is a genetically diverse assemblage of taxa and effective vector control requires a clear understanding of the geographic distribution and epidemiological importance of its taxa. The nuclear ribosomal internal transcribed spacer 2 (ITS-2) is frequently used to infer the systematics of triatomines. However, oftentimes amplification and sequencing of ITS-2 fails, likely due to both the large polymerase chain reaction (PCR) product and polymerase slippage near the 5' end. To overcome these challenges we have designed new primers that amplify only the 3'-most 200 base pairs of ITS-2. This region distinguishes the ITS-2 group for 100% of known T. dimidiata haplotypes. Furthermore, we have developed a PCR-restriction fragment length polymorphism (RFLP) approach to determine the ITS-2 group, greatly reducing, but not eliminating, the number of amplified products that need to be sequenced. Although there are limitations with this new PCR-RFLP approach, its use will help with understanding the geographic distribution of T. dimidiata taxa and can facilitate other studies characterising the taxa, e.g. their ecology, evolution and epidemiological importance, thus improving vector control.
Highlights
Previous technique and disadvantages - Four genetically distinct taxa of T. dimidiata have been identified in southern Mexico, Central America and northern South America by internal transcribed spacer 2 (ITS-2)
Using the primers designed by Marcilla et al (2001), the 900 bp ITS-2 fragment was successfully amplified from only 59.6% (199/334) of samples by polymerase chain reaction (PCR)
In silico analysis shows the PCR-restriction fragment length polymorphism (RFLP) assay using a sequential digest with the restriction enzyme BsrGI followed by ApoI successfully distinguishes all known ITS-2 group 3 haplotypes from groups 1A, 1B and 2 and distinguishes all of the group 2 haplotypes with exception of a rare group 2 variant
Summary
Previous technique and disadvantages - Four genetically distinct taxa of T. dimidiata have been identified in southern Mexico, Central America and northern South America by ITS-2 (groups 1A, 1B, 2 and 3). The primers most commonly used to determine ITS-2 taxa are complementary to the conserved, flanking regions of 5.8S and 28S rDNA and amplify a ~900 base pairs (bp) fragment containing the complete sequence of ITS-2 (Marcilla et al 2001). With these primers, oftentimes the template fails to amplify and/or produce interpretable sequence. Our approach is a less expensive and technically easier method for determining the ITS-2 group of T. dimidiata, so is amenable to endemic countries and provides much needed genetic information more quickly
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