Abstract

CHENA, L et al. Standardization of a PCR-RFLP technique based on mitochondrial cyt b gen as a tool to identify feeding sources of hematophagous insects. Mem. Inst. Investig. Cienc. Salud [online]. 2014, vol.12, n.2, pp.33-42. ISSN 1812-9528.

Identification of feeding sources of hematophagous insects can provide information about the vectorial capacity, feeding patterns in natural conditions and indirectly provide data on possible disease reservoirs preferences. Several identification techniques are used, most of them based on antigen-antibody reactions. Recently molecular assays have been developed and, some of these assays can detect and identificate only human blood. Other assays, like the polymerase chain reaction (PCR) based on mitochondrialcytochrome bgene, have shown high sensitivity and specificity allowing detection and identification of other vertebrate species. The aim of this study was to standardize a PCR-RFLP based on mitochondrial cytochrome bgene (cyt b) in order to determine blood meal from insects. Initially bioinformatic analysis was performed for searching and alignment of cyt bsequences of potential hosts available at the GenBank database. Blood samples from potential vertebrate hosts were used and PCR was performed using specific primers that amplify a region of cyt b gene. The products were digested with restriction enzymes (RFLP), generating specific electrophoresis patterns for several vertebrates.The PCR technique for cyt bgene was standardized allowing the detection of at least 1 µL of blood. The 359 bp band was correctly amplified and the profiles obtained after the enzyme digestion with HaeIII and MwoI were the expected for human, chicken, dog and rodents. These results showed the usefulness of the PCR-RFLP of cyt bgene that, with a single pair of primers followed digestion using two restriction enzymes, allowed the differentiation of the vertebrate species of our interest through the patterns obtained without sequencing having also the advantage of detecting small volumes of blood sample.

Keywords : PCR; RFLP; cytochrome b; feeding source; leishmaniasis.

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