High-Resolution Melting (HRM) of the Cytochrome B Gene: A Powerful Approach to Identify Blood-Meal Sources in Chagas Disease Vectors

Abstract

Methods to determine blood-meal sources of hematophagous Triatominae bugs (Chagas disease vectors) are serological or based on PCR employing species-specific primers or heteroduplex analysis, but these are expensive, inaccurate, or problematic when the insect has fed on more than one species. To solve those problems, we developed a technique based on HRM analysis of the mitochondrial gene cytochrome B (Cyt b). This technique recognized 14 species involved in several ecoepidemiological cycles of the transmission of Trypanosoma cruzi and it was suitable with DNA extracted from intestinal content and feces 30 days after feeding, revealing a resolution power that can display mixed feedings. Field samples were analyzed showing blood meal sources corresponding to domestic, peridomiciliary and sylvatic cycles. The technique only requires a single pair of primers that amplify the Cyt b gene in vertebrates and no other standardization, making it quick, easy, relatively inexpensive, and highly accurate.

Eco-Epidemiology of Chagas Disease in an Endemic Area of Colombia: Risk Factor Estimation, Trypanosoma cruzi Characterization and Identification of Blood-Meal Sources in Bugs

“…23 Reaction conditions and thermal profile were performed as described previously. 24 The standard species used in this study were opossum (Didelphis marsupialis), mouse (Mus musculus L.), cow (Bos taurus), goat (Capra aegagrus), dog (Canis lupus familiaris), sheep (Ovis aries), horse (Equus caballus), pig (Sus scrofa), rabbit (Oryctolagus cuniculus), cat (Felis catus), rat (Rattus norvegicus), donkey (Equus asinus), human (Homo sapiens L.), and chicken (Gallus gallus L.). The species were selected by DNA availability, closeness to human environments, and ecoepidemiological importance.…”

Eco-epidemiological study of an endemic Chagas disease region in northern Colombia reveals the importance of Triatoma maculata (Hemiptera: Reduviidae), dogs and Didelphis marsupialis in Trypanosoma cruzi maintenance

“…We carried out a cytochrome b gene high-resolution melting (HRM) analysis from insect faeces following standard procedures reported elsewhere [18, 29]. Briefly, the cyt b gene was amplified using the primers forward (5′-CCCCTCAGAATGATATTTGTCCTCA- 3′) and reverse (5′-CCATCCAACATCTCAGCATGATGAAA-3′) [30].…”

Rapid detection of Echinococcus species by a high-resolution melting (HRM) approach

“…For Trypanosoma cruzi, the HRM method has been implemented based on the amplification of a 383-bp DNA fragment of the cytochrome b gene (cyt b), allowing the effective differentiation of 14 genotypes and showing that the use of amplicons derived from mitochondrial genes is reliable and sufficiently robust for use in HRM [14]. …”