MutaREAL® NOROVIRUS Real Time RT-PCR KIT
INTENDED USE
The MutaREAL® Norovirus real time RT-PCR kit is a qualitative assay for the specific detection of Norovirus (GG I and II) in stool samples using real time RT-PCR capillary systems (e.g. Light Cycler® 1.5/ 2.0, Roche). The MutaREAL® Norovirus real time RT-PCR kit is licensed from Roche Molecular Systems, Inc.
INTRODUCTION
Non-bacterial Gastroenteritis is mainly caused by species of human-pathogen noroviruses. These high infectious viruses have a non-enveloped RNA genome and belong to the family of Caliciviridae. They are resistant against environmental influences, e.g. against temperatures between -20°C and +60°C, acids (pH3) and chlorite (10mg/ L). NV infections may occur the whole year but in Europe a seasonal increase from October - March is typical. NV transmission happens direct from person to person by contact-/ smear-infection or also indirect by contaminated food or water. Already a low dose of NV particles is sufficient to release epidemic infections. Therefore especially closed environments (hospitals, elderly homes, kindergartens, sport-courts, cruise-ships etc.) are affected. Positive tested persons have to be registered (RKI) and isolated for several days.
Due to their high genetic variability, human-pathogen NV is subdivided in different geno-groups/genotypes. But because MutaREAL® Norovirus reagents are routine up-dated using different sequence data bases, they recognize even strains from high genetic diversity, e.g.: GGI: Norwalk, Southampton, Queens Arms, Desert Shield, Winchester, Sindlesham, Chiba GGII: Lordsdale, Melksham, Hawaii, Mexico, Leeds, Hillingdon, Snow Mountain, Toronto, Bristol
PRINCIPLE OF THE TEST
MutaREAL® Norovirus real time RT-PCR kit
MutaREAL® Norovirus real time RT-PCR kit contains specific primers, fluorescence-marked probes and additional material for the detection of Norovirus (genogroup I and II). Human stool samples may be used as starting material for RNA-extraction and following pathogen analysis with MutaLREAL® Norovirus real time RT-PCR kit.
Reverse transcription (RT) of potentially contained viral RNA to cDNA and subsequent amplification of norovirus-specifc fragments by means of PCR is done in only one step. Target sequence for the detection is the region in the ORF1 / ORF2 - junction. Detection of NV amplificates is achieved in real time by hybridization and hydrolysis of NV-specific fluorescence probes. Their emitted signal is measured (during PCR process) by the optical unit of the real time PCR system in use (LightCycler 1.5 respectively 2.0- software). Norovirus-specific amplification is measured in the F1-channel (530 nm).
Furthermore, MutaREAL® Norovirus real time RT-PCR - kit contains also an additonal RNA internal control. This is detected in an independent (heterologous) amplification step. This internal control (IC) identifies potential inhibition of reverse transcription/ amplification during PCR. The internal control is measured at 710 nm in channel F3 (LightCycler 1.5) respectively F6 (LightCycler 2.0).
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