Abstract
BackgroundNorovirus 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.ResultsIn 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.ConclusionsThese standardized products may contribute to the reliable and accurate diagnosis by RT-PCR of norovirus outbreaks, when conducted by laboratories located in different regions.
Highlights
Norovirus is one of the most common causes of nonbacterial gastroenteritis in humans
Cloning of reverse transcription PCR (RT-PCR) amplified NoV, human rotavirus (HRV), and hepatitis A virus (HAV) genes Viral RNA extracted from the stool samples of patient hospitalized from viral diarrhea was used as the template for RT-PCR
These three NoV capsid genotypes were cloned into the pGEM-T Easy Vector, and the resulting plasmids were used as backbones for the construction of standardized positive control plasmids
Summary
Norovirus is one of the most common causes of nonbacterial gastroenteritis in humans. Detection of norovirus is a major public health concern. 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 falsenegative results due to PCR inhibitors and suboptimal reaction conditions. Gastroenteritis, known as “stomach flu”, is a major public health concern and causes over 1.8 million deaths worldwide every year in children younger than five. Several studies and diagnostic analyses have shown that noroviruses (NoVs) are the leading cause of acute nonbacterial gastroenteritis. The gastroenteritis caused by NoVs is mild and of short duration, it affects persons of all ages and sometimes leads to death.
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