Standardized positive controls for detection of norovirus by reverse transcription PCR

Norovirus is one of the most common causes of nonbacterial gastroenteritis in humans. Rapid spread by contaminated food and person-to-person transmission through the fecal-oral route are characteristics of norovirus epidemiology and result in high morbidity in vulnerable patient populations. Therefore, detection of norovirus is a major public health concern. Currently, the most common method for detecting and differentiating among norovirus strains in clinical and environmental samples is reverse transcription PCR (RT-PCR). Standardized positive controls used in RT-PCR assays to detect norovirus are designed to overcome the problem of false-negative results due to PCR inhibitors and suboptimal reaction conditions.

In the current study, four types of RNA transcripts were produced from plasmids: norovirus GI-5 and GII-4 capsid regions with human rotavirus (VP7 gene derived) fragment insertions, and norovirus GI-6 and GII-4 capsid regions with hepatitis A virus (VP1/P2A gene derived) fragment insertions. These size-distinguishable products were used as positive controls under the RT-PCR assay conditions used to detect NoV in stool and groundwater samples. Their reliability and reproducibility was confirmed by multiple sets of experiments.

These standardized products may contribute to the reliable and accurate diagnosis by RT-PCR of norovirus outbreaks, when conducted by laboratories located in different regions.