Novel polymerase chain reaction-restriction fragment length polymorphism assay to determine internal transcribed spacer-2 group in the Chagas disease vector,Triatoma dimidiata (Latreille, 1811)

Triatoma dimidiata is the most important Chagas disease insectvector in Central America as this species is primarily responsible forTrypanosoma cruzi transmission to humans, the protozoanparasite that causes Chagas disease. T. dimidiata sensu lato isa genetically diverse assemblage of taxa and effective vector control requires aclear understanding of the geographic distribution and epidemiologicalimportance 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 thelarge polymerase chain reaction (PCR) product and polymerase slippage near the5'' end. To overcome these challenges we have designed new primers that amplifyonly the 3''-most 200 base pairs of ITS-2. This region distinguishes the ITS-2group for 100% of known T. dimidiata haplotypes. Furthermore,we have developed a PCR-restriction fragment length polymorphism (RFLP) approachto determine the ITS-2 group, greatly reducing, but not eliminating, the numberof amplified products that need to be sequenced. Although there are limitationswith this new PCR-RFLP approach, its use will help with understanding thegeographic distribution of T. dimidiata taxa and can facilitateother studies characterising the taxa, e.g. their ecology, evolution andepidemiological importance, thus improving vector control.