Detection and characterization of norovirus outbreaks in Germany: application of a one-tube RT-PCR using a fluorogenic real-time detection system.
Outbreaks of gastroenteritis caused by Norovirus are an increasing public health problem worldwide. The virus is transmitted by contaminated food and by person-to-person. In Germany, a new health reporting system including Norovirus infections has been introduced in 2001. Norovirus outbreaks (73%) diagnosed in our lab occurred in the first half of the year. Phylogenetic analysis shows that 90% belonged to genogroup II (GGII) with the majority related to prototypes Lordsdale/93 and Tarragona/2001. To date, PCR is the most sensitive and specific method for the detection but several procedures are needed to evaluate the amplification product. To minimize the risk of product carryover, a fast and simple procedure with a minimal number of steps are required. A one-tube RT-PCR method is described using the real-time TaqMan-PCR system. Primer sets and probes for both genogroup I (GGI) and genogroup II (GGII) sequences were developed. The specificity and sensitivity of this method was evaluated on 70 Norovirus positive stool samples of 70 outbreaks in Germany and 34 European samples by comparing the detection rate to those of an in-house RT-nested PCR. Overall, 93% of the PCR positive samples have been detected by the TaqMan-PCR including isolates of four different GGI and seven different GGII genotypes. Using plasmid standards for quantitation, virus loads between 10(2) and 10(10) genomic equivalents/ml stool suspension were found. The advantages of the one-step RT/TaqMan-PCR system for detection and quantitation of Norovirus are the high throughput of clinical samples and a decrease of the risk of contamination.
Höhne
M
M
Department of Molecular Epidemiology, of Viral Pathogens, Robert Koch-Institute, Berlin, Germany. hoehnem@rki.de
Schreier
E
E
D017250:Caliciviridae Infections / Q000175:diagnosis / Q000453:epidemiology* / Q000821:virology; D004196:Disease Outbreaks; D005243:Feces / Q000821:virology; D005456:Fluorescent Dyes; D005759:Gastroenteritis / Q000175:diagnosis / Q000453:epidemiology* / Q000821:virology; D005838:Genotype; D005858:Germany / Q000453:epidemiology; D029322:Norovirus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D010802:Phylogeny; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D019914:Taq Polymerase; D019562:Viral Load
D005456:Fluorescent Dyes; D019914:Taq Polymerase