Article thumbnail
Location of Repository

Fast detection of Noroviruses using a real-time PCR assay and automated sample preparation

By Michael Schmid, Rainer Oehme, Gunnar Schalasta, Stefan Brockmann, Peter Kimmig and Gisela Enders

Abstract

BACKGROUND: 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. METHODS: We have designed a new real-time RT-PCR assay on the LightCycler (LC) with SYBR Green detection and melting curve analysis (T(m)) 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. RESULTS: 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. CONCLUSIONS: 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: Technical Advance
Publisher: BioMed Central
Year: 2004
DOI identifier: 10.1186/1471-2334-4-15
OAI identifier: oai:pubmedcentral.nih.gov:434506
Provided by: PubMed Central

Suggested articles

Citations

  1. (1996). Atmar RL: Evaluation of a degenerate primer for the PCR detection of human caliciviruses. Arch Virol
  2. (1997). Atmar RL: Use of heat release and an internal RNA standard control in reverse transcriptionPCR detection of Norwalk virus from stool samples.
  3. (2003). Broadly reactive and highly sensitive assay for Norwal.like viruses based on real-time quantitative reverse transcription-PCR.
  4. (1972). Chanock RM: Visualization by immune electron microscopy of a 27-nm particle associated with a non-bacterial gastroenteritis.
  5. (2002). Detection and differentiation of Norwalk virus by reverse transcription polymerase chain reaction and enzyme-linked immunosorbent assay.
  6. (1996). Detection of sporadic cases of Norwalk-like virus (NLV) and astrovirus infection in a single Irish hospital from
  7. (2003). DW: Evaluation of a commercial ELISA for detecting Norwalklike virus antigen in faeces.
  8. (1995). DWG: Broadly reactive reverse transcriptase polymerase chain reaction (RT-PCR) for the diagnosis of SRSV-associated gastroenteritis.
  9. (1995). DWG: Detection of small round structured viruses in shellfish by reverse transcription-PCR. Appl Environ Microbiol
  10. (1993). DWG: Norwalklike viruses: demonstration of genomic diversity by polymerase chain reaction.
  11. (1992). Estes MK: Detection of Norwalk virus in stool by polymerase chain reaction.
  12. (2001). Estes MK: Diagnosis of noncultivatable gastroenteritis viruses, the human caliciviruses. Clin Microbiol Rev
  13. (1994). Estes MK: Norwalk virus infection of volunteers: new insights based on improved assays.
  14. (1994). Estes MK: Sequence diversity of small, round-structured viruses in the Norwalk virus group.
  15. (2002). Food- and waterborne disease outbreaks in Germany: three years experience (1999–2001) in Baden-Württemberg.
  16. (2000). Frankhauser RL: Genetic classification of "Norwalk-like viruses".
  17. (1994). Glass RI: Application of PCR to detect Norwalk virus in fecal specimens from outbreaks of gastroenteritis.
  18. (2003). HW: Laboratory diagnosis of norovirus: which method is best? Intervirology
  19. (2001). Kapikian AZ: Human Caliciviruses.
  20. (2000). Künkel U: Molecular epidemiology of outbreaks of gastroenteritis associated with small round structured viruses in Germany in 1997/98. Arch Virol
  21. (2003). MP: International collaborative study to compare reverse transcriptase PCR assays for detection and genotyping of norovirus.
  22. (2000). MPG: Genetic polymorphism across regions of the three open reading frames of "Norwalk-like viruses". Arch Virol
  23. (1997). MPG: The incidence and genetic variability of small round-structured viruses in outbreaks of gastroenteritis in the Netherlands. J Infect Dis
  24. (1990). Rapid and simple method for purification of nucleic acids.
  25. (2000). RE: Evidence for airborne transmission of Norwalk-like virus (NLV) in a hotel restaurant. Epidemiol Infect
  26. (1995). RI: Detection and differentiation of antigenically distinct small roundstructured viruses (Norwalk-like viruses) by reverse transcription-PCR and Southern hybridisation.
  27. (1998). RI: Molecular epidemiology of „Norwalk-like viruses" in outbreaks of gastroenteritis in The United States. J Infect Dis
  28. (1999). Silvennoinen E, von Bonsdorff CH: Outbreak of viral gastroenteritis due to drinking water contaminated by Norwalk-like viruses.
  29. (2000). Verfolgung von Gruppenerkrankungen mit Norwalk-like Viren (NLV) in BadenWürttemberg. Gesundheitswesen
  30. (2003). Viral gastroenteritis outbreaks

To submit an update or takedown request for this paper, please submit an Update/Correction/Removal Request.