Development of a one step real-time RT-PCR method for sensitive detection of human astrovirus.
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.
Royuela
Enrique
E
Servicio de Virología, Centro Nacional de Microbiología, Instituto de Salud Carlos III, Ctr. Majadahonda-Pozuelo km 2, 28220 Majadahonda, Madrid, Spain.
Negredo
Ana
A
Sánchez-Fauquier
Alicia
A
D000208:Acute Disease; D019350:Astroviridae Infections / Q000175:diagnosis* / Q000453:epidemiology*; D052160:Benzothiazoles; D002675:Child, Preschool; D003959:Diamines; D005240:Feasibility Studies; D005456:Fluorescent Dyes; D005759:Gastroenteritis / Q000175:diagnosis / Q000453:epidemiology / Q000821:virology; D006801:Humans; D015994:Incidence; D007201:Indicator Dilution Techniques; D001257:Mamastrovirus / Q000145:classification / Q000302:isolation & purification*; D009691:Nucleic Acid Denaturation; D009930:Organic Chemicals; D011804:Quinolines; D012367:RNA, Viral / Q000032:analysis* / Q000235:genetics; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D016036:Seroepidemiologic Studies; D012703:Serotyping; D013696:Temperature
D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; D012367:RNA, Viral; C098022:SYBR Green I