Molecular detection of dengue viruses in field caught Aedes aegypti mosquitoes from northeastern Argentina.
Most molecular protocols for Dengue virus detection described so far are time consuming and cumbersome with mosquito samples. In order to count with a sensitive and specific molecular detection system for monitoring possible Dengue outbreaks and circulating viral serotypes in field-caught Aedes aegypti populations from Northeastern Argentina, a RT-PCR and RFLP assay was developed. The original RT-PCR assay proposed by Sudiro et al. for human serum was optimized for mosquito samples. Modifications were done at the RNA extraction-purification and at the thermal profile steps. The generic 230 bp amplicon was validated by RFLP assay and cycle sequencing. Results showed that, due to the generic characteristic of the primers used, certain mosquito genome regions could be co-amplified, making confirmation of the Dengue specific amplicon by RFLP assay a required step. Under these conditions, the proposed method can be employed as a Dengue viral generic screening procedure in Aedes aegypti mosquito samples, giving in our hands an estimated 99.52% of confirmed negatives (207/208 tested samples).
Liotta
Domingo Javier
DJ
Laboratorio de Biología Molecular Aplicada, Facultad de Ciencias Exactas, Químicas y Naturales, Universidad Nacional de Misiones, Argentina. liotta@mbf.unam.edu.ar
Cabanne
Gustavo
G
Campos
Rodolfo
R
Tonon
Sergio Andrés
SA
D000330:Aedes / Q000821:virology*; D000818:Animals; D001118:Argentina / Q000453:epidemiology; D001483:Base Sequence; D003715:Dengue / Q000453:epidemiology / Q000821:virology*; D003716:Dengue Virus / Q000302:isolation & purification*; D005260:Female; D006801:Humans; D007303:Insect Vectors / Q000821:virology*; D008969:Molecular Sequence Data; D012150:Polymorphism, Restriction Fragment Length; D020133:Reverse Transcriptase Polymerase Chain Reaction; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid