Published in:
Open Access 01-12-2004 | Technical advance
Fast detection of Noroviruses using a real-time PCR assay and automated sample preparation
Published in: BMC Infectious Diseases | Issue 1/2004
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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 (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.
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.