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Diagnostic Methods

Diagnostic methods for norovirus focus on detecting viral RNA or viral antigen. Most public health and clinical virology laboratories test for norovirus by using real-time reverse transcription-polymerase chain reaction (RT-qPCR) assays.

RT-qPCR Assays

RT-qPCR assays are the preferred laboratory method for detecting norovirus. These assays are very sensitive and can detect as few as 10 to 100 norovirus copies per reaction. They use different oligonucleotide primer sets to differentiate genogroup I and genogroup II norovirus. RT-qPCR assays are also quantitative and can provide estimates of viral load. The assays may be used to detect norovirus in stool, vomitus, foods, water, and environmental specimens.

Recently, several commercial molecular assays designed to simultaneously detect multiple gastrointestinal pathogens, including norovirus, have become available. The sensitivity of these assays for norovirus is in the same range as RT-qPCR.

Enzyme Immunoassays

Rapid commercial assays, such as enzyme immunoassays (EIAs), detect norovirus antigen but these kits have poor sensitivity (50-75%) and are not recommended for diagnosing norovirus infection in sporadic cases of gastroenteritis. The RIDASCREEN Norovirus 3rd Generation EIA has been cleared by Food and Drug Administration for preliminary identification of norovirus when testing multiple specimens during outbreaks. However, samples that test negative should be confirmed by a second technique, such as RT-qPCR. Thus, EIA kits should not replace molecular methods during outbreak investigations.

Conventional RT-PCR Assays for Genotyping

Conventional RT-PCR followed by sequence analysis of the RT-PCR products is used for norovirus genotyping. Dual typing for norovirus is being implemented by laboratories participating in CaliciNet, a national laboratory surveillance network for norovirus outbreaks coordinated by CDC. This consist of RT-PCR amplification a partial region of both the polymerase gene and the capsid gene (region B-C) in a single reaction for either genogroup I or genogroup II viruses. The sequences obtained are compared to CaliciNet reference sequences for typing. An example of dual typing is the GII.P16-GII.4 Sydney norovirus strain. Dual typed information is now included on CDC’s CaliciNet data page.