sybr-green-i and Gastroenteritis

Topics: Animals; Benzothiazoles; Diamines; Diarrhea; Dog Diseases; Dogs; Feces; Gastroenteritis; Kobuvirus; Picornaviridae Infections; Quinolines; Real-Time Polymerase Chain Reaction; Sensitivity and Specificity

A novel SYBR Green based real-time RT-PCR assay for detection of genogroup III bovine noroviruses (BoNoV) was developed and the assay applied to 419 faecal samples from calves with and without diarrhoea. The samples were obtained from 190 Norwegian dairy and beef herds. BoNoV was detected in 49.6% of the samples from 61.1% of the herds indicating that BoNoV is ubiquitous in Norway. The overall prevalence was not significantly different in diarrhoea and non-diarrhoea samples. Analyses of polymerase gene sequences revealed both genotype III/1 and III/2 with genotype III/2 (Newbury2-like) being the most prevalent. Detected capsid sequences were restricted to Newbury2-like and the chimeric Bo/Thirsk10/00/UK strain. The RNA polymerase genotypes of the circulating BoNoVs in Norway were predicted by melting temperature analysis. Additional data from a challenge experiment suggest that a high proportion of young calves are shedding low levels of BoNoV for a prolonged time after recovering from the associated diarrhoea. The findings may explain some of the discrepancies in detection rates from previous studies and explain why some studies have failed to detect significant prevalence differences between calves with and without diarrhoea. It may also shed new light on some epidemiological aspects of norovirus infections.

Topics: Animals; Benzothiazoles; Caliciviridae Infections; Cattle; Cattle Diseases; Diamines; Feces; Gastroenteritis; Genotype; Molecular Epidemiology; Molecular Sequence Data; Norovirus; Norway; Organic Chemicals; Prevalence; Quinolines; Reverse Transcriptase Polymerase Chain Reaction; RNA, Viral; Sequence Analysis, DNA; Staining and Labeling; Virology

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

Topics: Benzothiazoles; Caliciviridae Infections; Community-Acquired Infections; Cross Infection; Diamines; Disease Outbreaks; Gastroenteritis; Humans; Norovirus; Organic Chemicals; Polymerase Chain Reaction; Quinolines; Taq Polymerase

A multiplex real-time RT-PCR protocol for the simultaneous detection of noroviruses ("Norwalk-like viruses") of genogroups I and II, human astroviruses and enteroviruses is described. The protocol was developed and evaluated using the LightCycler and corresponding SYBR Green reagents. New primers were designed within conserved genome regions to optimize the detection range of virus subtypes of each genus. To enable the development of a multiplex PCR assay within one tube (capillary), similar mastermix- and cycling-conditions were respected for each individual primer system. Subsequent melting curve analysis allowed the determination of possible dual-contaminations of entero- and noro- or astroviruses by the formation of dual peaks. Special care was taken to minimize the loss of sensitivity, since the detection of small viral contaminations is a crucial parameter especially for food analysis. The multiplex assay was compared successfully to the single SYBR Green assay, and revealed to be at least 10 times more sensitive than the one obtained with an endpoint PCR thermocycler protocol published previously.

Topics: Benzothiazoles; Diamines; Enterovirus; Gastroenteritis; Humans; Mamastrovirus; Norwalk virus; Organic Chemicals; Quinolines; Reverse Transcriptase Polymerase Chain Reaction; RNA, Viral

Noroviruses (NoV) have become one of the most commonly reported causative agents of large outbreaks of non-bacterial acute gastroenteritis worldwide as well as sporadic gastroenteritis in the community. Currently, reverse transcriptase polymerase chain reaction (RT-PCR) assays have been implemented in NoV diagnosis, but improvements that simplify and standardize sample preparation, amplification, and detection will be further needed. The combination of automated sample preparation and real-time PCR offers such refinements.. We have designed a new real-time RT-PCR assay on the LightCycler (LC) with SYBR Green detection and melting curve analysis (Tm) to detect NoV RNA in patient stool samples. The performance of the real-time PCR assay was compared with that obtained in parallel with a commercially available enzyme immunoassay (ELISA) for antigen detection by testing a panel of 52 stool samples. Additionally, in a collaborative study with the Baden-Wuerttemberg State Health office, Stuttgart (Germany) the real-time PCR results were blindly assessed using a previously well-established nested PCR (nPCR) as the reference method, since PCR-based techniques are now considered as the "gold standard" for NoV detection in stool specimens.. Analysis of 52 clinical stool samples by real-time PCR yielded results that were consistent with reference nPCR results, while marked differences between the two PCR-based methods and antigen ELISA were observed. Our results indicate that PCR-based procedures are more sensitive and specific than antigen ELISA for detecting NoV in stool specimens.. The combination of automated sample preparation and real-time PCR provided reliable diagnostic results in less time than conventional RT-PCR assays. These benefits make it a valuable tool for routine laboratory practice especially in terms of rapid and appropriate outbreak-control measures in health-care facilities and other settings.

Topics: Adolescent; Adult; Benzothiazoles; Caliciviridae Infections; Child; Child, Preschool; Diamines; Enzyme-Linked Immunosorbent Assay; Feces; Fluorescent Dyes; Gastroenteritis; Humans; Middle Aged; Norovirus; Organic Chemicals; Polymerase Chain Reaction; Quinolines; Reproducibility of Results; Reverse Transcriptase Polymerase Chain Reaction; RNA, Viral; Sensitivity and Specificity

We developed an assay for the detection and quantitation of norovirus with the LightCycler SYBR Green-based real-time reverse transcription-PCR (real-time LC RT-PCR) and previously published primers in the capsid and the polymerase gene. One hundred thirty-two stool specimens from the Provincial Laboratory for Public Health (Microbiology), Alberta, Canada, and the Centers for Disease Control and Prevention, Atlanta, Ga., were used to validate the new assay. The samples were collected from patients involved in outbreaks of acute gastroenteritis or children who presented with sporadic gastroenteritis. The real-time LC RT-PCR assay detected norovirus strains from three genogroup I (G-I) clusters (G-I/1, G-I/2, and G-I/3) and 10 genogroup II (G-II) clusters (G-II/1, G-II/2, G-II/3, G-II/4, G-II/6, G-II/7, G-II/10, G-II/12, G-II/15, and G-II/16). There was 100% concordance with the results from 58 stool specimens which tested positive by conventional RT-PCR assays. By dilution analysis, the real-time LC RT-PCR was 10,000 times more sensitive than the conventional RT-PCR. The new assay increased the number of samples in which noroviruses were detected by 19%. The real-time LC RT-PCR had a wide dynamic range, detecting from 5 to 5 x 10(6) copies of RNA per reaction, resulting in a theoretical lower limit of detection of 25,000 copies of RNA per g of stool. No cross-reactions were found with specimens containing sapovirus, rotavirus, astrovirus, and adenovirus. Because of the high sensitivity and specificity of the assay with a relatively rapid and simple procedure, the real-time LC RT-PCR will be useful as a routine assay for the clinical diagnosis of norovirus infection.

Topics: Benzothiazoles; Caliciviridae Infections; Child; Child, Preschool; Diamines; Feces; Gastroenteritis; Hot Temperature; Humans; Norovirus; Organic Chemicals; Quinolines; Reverse Transcriptase Polymerase Chain Reaction; RNA, Viral; Sensitivity and Specificity