Molecular serotyping of dengue viruses in field-caught Aedes mosquitos by in-house RNA extraction/RT-PCR reagent kits.
Southeast Asian J Trop Med Public Health
;
2002 ; 33 Suppl 3(): 139-44
Artigo
em Inglês
| IMSEAR
| ID: sea-35815
ABSTRACT
We developed in-house RNA extraction and RT-PCR reagent kits for the molecular serotyping of dengue viruses in field-caught Aedes mosquitos. Mosquitos that showed positive results by ELISA or IFA were selected for the identification of dengue viruses in order to predict the distribution of the four dengue serotypes. Total RNA was extracted from one whole mosquito as well as from one dissected mosquito by our in-house RNA extraction reagents using the modified method of guanidinium thiocyanate denaturation and isopropanol precipitation. The extracted RNA was amplified by our in-house RT-PCR reagents specific for each dengue serotype under optimized conditions. Dengue viral RNA extracted from a single mosquito as well as from the head and thorax of one dissected mosquito could be detected successfully; it could not be found in the abdomen, however. These results indicated that most of the dengue viruses were located in the head and thorax rather than in the abdomen. The results of dengue serotyping showed a pure specific PCR product for each dengue serotype at 490, 230, 320 and 398 bp for DEN-1, DEN-2, DEN-3, and DEN-4 respectively. In addition, the detection sensitivity was very high an amount of RNA template equivalent to approximately 1/80 of a single mosquito could be detected by agarose gel electrophoresis and ethidium bromide staining. The coupling of RT-PCR-based surveillance of dengue viral infection with disease mapping data (Geograpical Information System, GIS) could serve as an alternative epidemiological means of providing an early warning of dengue fever/dengue hemorrhagic fever epidemics.
Texto completo:
DisponíveL
Índice:
IMSEAR (Sudeste Asiático)
Assunto principal:
RNA
/
Sensibilidade e Especificidade
/
Reação em Cadeia da Polimerase Via Transcriptase Reversa
/
Vírus da Dengue
/
Habitação
/
Indicadores e Reagentes
/
Animais
Tipo de estudo:
Estudo diagnóstico
Idioma:
Inglês
Revista:
Southeast Asian J Trop Med Public Health
Ano de publicação:
2002
Tipo de documento:
Artigo
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