Fast detection of Noroviruses using a real-time PCR assay and automated sample preparation.
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.
Schmid
Michael
M
Institute for Virology, Infectiology and Epidemiology e,V, 70193 Stuttgart, Germany. schmid@labor-enders.de
Oehme
Rainer
R
Schalasta
Gunnar
G
Brockmann
Stefan
S
Kimmig
Peter
P
Enders
Gisela
G
D000293:Adolescent; D000328:Adult; D052160:Benzothiazoles; D017250:Caliciviridae Infections / Q000175:diagnosis* / Q000821:virology; D002648:Child; D002675:Child, Preschool; D003959:Diamines; D004797:Enzyme-Linked Immunosorbent Assay; D005243:Feces / Q000821:virology*; D005456:Fluorescent Dyes; D005759:Gastroenteritis / Q000175:diagnosis* / Q000821:virology; D006801:Humans; D008875:Middle Aged; D029322:Norovirus / Q000235:genetics / Q000276:immunology / Q000302:isolation & purification*; D009930:Organic Chemicals; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D011804:Quinolines; D012367:RNA, Viral / Q000032:analysis*; D015203:Reproducibility of Results; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012680:Sensitivity and Specificity
D052160:Benzothiazoles; D003959:Diamines; D005456:Fluorescent Dyes; D009930:Organic Chemicals; D011804:Quinolines; D012367:RNA, Viral; C098022:SYBR Green I