RESUMO
The Anopheles gambiae sensu lato species complex consists of a number of cryptic species with different habitats and behaviours. These morphologically indistinct species are identified by chromosome banding. Several molecular diagnostic techniques for distinguishing between An. coluzzii and An. gambiae are still under improvement. Although, the current SINE method for identification between An. coluzzii and An. gambiae works reliably, this study describes a refinement of the SINE method to increase sensitivity for identification of An. coluzzii, An. gambiae and An. arabiensis based on amplicon dissociation curve characteristics. Field-collected samples, laboratory-reared colonies and crossed specimens of the two species were used for the design of the protocol. An. gambiae, An. coluzzii, and hybrids of the two species were sampled from Ghana and An. arabiensis from Kenya. Samples were first characterised using conventional SINE PCR method, and further assayed using SYBR green, an intercalating fluorescent dye. The three species and hybrids were clearly differentiated using the melting temperature of the dissociation curves, with derivative peaks at 72°C for An. arabiensis, 75°C for An. gambiae and 86°C for An. coluzzii. The hybrids (An. gambiae / An. coluzzii) showed both peaks. This work is the first to describe a SYBR green real time PCR method for the characterization of An. arabiensis, An. gambiae and An. coluzzii and was purposely designed for basic melt-curve analysis (rather than high-resolution melt-curve) to allow it to be used on a wide range of real-time PCR machines.
Assuntos
Anopheles/genética , Malária/genética , Técnicas de Diagnóstico Molecular/métodos , Mosquitos Vetores/genética , Reação em Cadeia da Polimerase/métodos , Animais , Anopheles/classificação , Anopheles/parasitologia , Benzotiazóis , DNA/química , DNA/genética , Diaminas , Corantes Fluorescentes/química , Gana , Quênia , Malária/diagnóstico , Malária/parasitologia , Mosquitos Vetores/parasitologia , Compostos Orgânicos/química , Quinolinas , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Especificidade da EspécieRESUMO
BACKGROUND: The increasing spread of insecticide resistance in malaria vectors has been well documented across sub-Saharan Africa countries. The influence of irrigation on increasing vector resistance is poorly understood, and is critical to successful and ethical implementation of food security policies. This study investigated the insecticide resistance status of An. gambiae (s.l.) mosquitoes collected from the irrigated rice area of Okyereko, a village containing about 42 hectares of irrigated field within an irrigation project plan in the Central Region of Ghana. Large amounts of insecticides, herbicides and fertilizers are commonly used in the area to boost the annual production of the rice. METHODS: Mosquito larvae were collected and adults were assayed from the F1 progeny. The resistance status, allele and genotype were characterized using WHO susceptibility testing and PCR methods respectively. RESULTS: The An. gambiae (s.l.) populations from Okyereko are highly resistant to DDT and pyrethroid insecticides, with possible involvement of metabolic mechanisms including the elevation of P450 and GST enzyme as well as P-gp activity. The population was mostly composed of An. coluzzii specimens (more than 96 %) with kdr and ace-1 frequencies of 0.9 and 0.2 %, respectively. CONCLUSION: This study brings additional information on insecticide resistance and the characterization of An. gambiae (s.l.) mosquitoes from Okyereko, which can be helpful in decision making for vector control programmes in the region.
