Development of a one step real-time RT-PCR method for sensitive detection of human astrovirus.
J Virol Methods
; 133(1): 14-9, 2006 Apr.
Article
em En
| MEDLINE
| ID: mdl-16321452
ABSTRACT
Human astrovirus (HAstV) has been recognized as the second most common cause of diarrhoea among children under 5 years old. To date, the true incidence of HAstV was underestimated when using enzyme immunoabsorbent assays (EIAs) and conventional reverse transcription (RT)-polymerase chain reaction (PCR) methods. The sensitivity of detection of EIA is insufficient and, although RT-PCR is more sensitive than EIA, the time required is a limitation for astrovirus detection. The aim of the study was to develop a real-time RT-PCR method in order to increase the sensitivity, to quantify the viral load and to minimize the time required for HAstV detection. The real-time RT-PCR reported here requires only one rapid step to obtain a high sensitivity (0.0052 infectious units (IU) (0.0026 IU/microl)) in all human astrovirus detected. The real-time RT-PCR detected IUs down to a 10(-6) dilution with an improvement in the detection limit of factor 10(4), whereas the conventional RT-PCR detected down to IUs 10(-2) dilution. This process is able to reduce the time of the assay and avoids the risk of contamination. The method described below has been validated with a panel of 100 clinical samples and the results obtained confirmed the high specificity of the assay; consequently, the application of this assay for molecular diagnosis is feasible as a versatile tool for ascertaining the true implication of HAstV in acute viral gastroenteritis.
Buscar no Google
Base de dados:
MEDLINE
Assunto principal:
Mamastrovirus
/
RNA Viral
/
Infecções por Astroviridae
/
Reação em Cadeia da Polimerase Via Transcriptase Reversa
Tipo de estudo:
Diagnostic_studies
/
Incidence_studies
/
Prognostic_studies
Limite:
Child, preschool
/
Humans
Idioma:
En
Ano de publicação:
2006
Tipo de documento:
Article