sybr-green-i and Astroviridae-Infections

Topics: Animals; Astroviridae Infections; Avastrovirus; Benzothiazoles; Diamines; Geese; Parvoviridae Infections; Parvovirinae; Quinolines; Real-Time Polymerase Chain Reaction; Reference Standards; Reproducibility of Results; Sensitivity and Specificity

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.

Topics: Acute Disease; Astroviridae Infections; Benzothiazoles; Child, Preschool; Diamines; Feasibility Studies; Fluorescent Dyes; Gastroenteritis; Humans; Incidence; Indicator Dilution Techniques; Mamastrovirus; Nucleic Acid Denaturation; Organic Chemicals; Quinolines; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; RNA, Viral; Sensitivity and Specificity; Seroepidemiologic Studies; Serotyping; Temperature

Human astroviruses (HAstVs) cause gastroenteritis. Real-time, reverse-transcription-polymerase chain reaction (RT2-PCR) was developed to quantitate HAstV RNA. An 88 bp amplicon from the conserved 3' genomic region was detected by binding of SYBR Green. RT2-PCR was reproducible, with a correlation coefficient of 0.998-1.00 and PCR efficiency of 94.4-100% (mean 97%). The coefficient of variation was 0.6-2.5%, dynamic range with RNA standard up to 5 x 10(8) RNA copies (RNACN) and sensitivity 5 RNACN. Of 54 blinded, archived stool samples from children hospitalized because of gastroenteritis tested by RT-PCR, 49 (91%) agreed by RT2-PCR for HAstV-positivity (Cohen kappa=0.81, 95%CI 0.66-0.97). HAstV RNACN in stools ranged from 7.6 x 10(1) to 3.6 x 10(14)copies/0.1g. Children coinfected with rotavirus had lower RNACN (mean log 4.22/standard deviation=2.26) than those without coinfection (7.57/3.06; p=.019). Children taking infant formula also had lower RNACN (5.96/2.98) than breast-fed or weaned children (8.73/2.92; p=.027). Higher RNACN tended to occur with longer duration of diarrhea for the episode (r=0.49, p=.064), but was not associated with change in age, gender, illness day, severity or breast-feeding. RT2-PCR quantitated HAstV RNA and RNACN in stool correlates with features of clinical illness.

Topics: Astroviridae Infections; Benzothiazoles; Caco-2 Cells; Child; Colony Count, Microbial; Diamines; Humans; Mamastrovirus; Organic Chemicals; Quinolines; Reverse Transcriptase Polymerase Chain Reaction; RNA, Viral; Rotavirus Infections