Abstract
BackgroundNoroviruses are the most common cause of non-bacterial gastroenteritis. Improved detection methods have seen a large increase in the number of human NoV genotypes in the last ten years. The objective of this study was to develop a fast method to detect, quantify and genotype positive NoV samples from Irish hospitals.ResultsA real-time RT-PCR assay and a Reverse Line Blot Hybridisation assay were developed based on the ORF1-ORF2 region. The sensitivity and reactivity of the two assays used was validated using a reference stool panel containing 14 NoV genotypes. The assays were then used to investigate two outbreaks of gastroenteritis in two Irish hospitals. 56 samples were screened for NoV using a real-time RT-PCR assay and 26 samples were found to be positive. Genotyping of these positive samples found that all positives belonged to the GII/4 variant of NoV.ConclusionThe combination of the Real-time assay and the reverse line blot hybridisation assay provided a fast and accurate method to investigate a NoV associated outbreak. It was concluded that the predominant genotype circulating in these Irish hospitals was GII/4 which has been associated with the majority of NoV outbreaks worldwide. The assays developed in this study are useful tools for investigating NoV infection.
Highlights
Noroviruses are the most common cause of non-bacterial gastroenteritis
We describe a real-time Reverse Transcription Polymerase Chain Reaction (RT-PCR) assay based on SYBR Green chemistry utilising a broadly reactive pair of primers for both Genogroup I (GI) and Genogroup II (GII) NoV based on the highly conserved ORF1-ORF2 junction
Development and validation of a SYBR green based RealTime Reverse Transcription (RT)-PCR assay for NoV Two degenerate reverse primers G1NVR and G2NVR (Table 1) were designed based on multiple alignments of 30 Genogroup I sequences and 120 Genogroup II sequences
Summary
Noroviruses are the most common cause of non-bacterial gastroenteritis. Improved detection methods have seen a large increase in the number of human NoV genotypes in the last ten years. The objective of this study was to develop a fast method to detect, quantify and genotype positive NoV samples from Irish hospitals. Noroviruses (NoV) are one of the most common causes of acute non-bacterial gastroenteritis in humans. Known as "Norwalk virus", NoV was first recognised in October 1968 in an elementary school in Norwalk, Ohio [1]. It is highly contagious and can be transmitted as an aerosol, through direct contact or via the faecal oral route causing explosive outbreaks in environments where people are in close contact such as hospitals, retirement homes, cruise ships, army bases, hotels and holiday resorts [2,3,4,5]. ORF1 encodes the nonstructural proteins, ORF2 encodes the major capsid protein VP1 and ORF3 encodes a minor structural protein VP2
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