Abstract
Noroviruses (NoVs) have high levels of genetic sequence diversities, which lead to difficulties in designing robust universal primers to efficiently amplify specific viral genomes for molecular analysis. We here described the practicality of sequence-independent amplification combined with DNA microarray analysis for simultaneous detection and genotyping of human NoVs in fecal specimens. We showed that single primer isothermal linear amplification (Ribo-SPIA) of genogroup I (GI) and genogroup II (GII) NoVs could be run through the same amplification protocol without the need to design and use any virus-specific primers. Related virus could be subtyped by the unique pattern of hybridization with the amplified product to the microarray. By testing 22 clinical fecal specimens obtained from acute gastroenteritis cases as blinded samples, 2 were GI positive and 18 were GII positive as well as 2 negative for NoVs. A NoV GII positive specimen was also identified as having co-occurrence of hepatitis A virus. The study showed that there was 100 % concordance for positive NoV detection at genogroup level between the results of Ribo-SPIA/microarray and the phylogenetic analysis of viral sequences of the capsid gene. In addition, 85 % genotype agreement was observed for the new assay compared to the results of phylogenetic analysis.
Highlights
Noroviruses (NoVs) are recognized as the leading causative agents of outbreaks and sporadic cases of nonbacterial acute gastroenteritis across all ages in humans, resulting in more than 267,000,000 annual infections worldwide and over 200,000 deaths each year among children under 5 years old in developing countries (Noel et al 1999; Patel et al 2008; Donaldson et al 2008)
Reverse transcription followed by PCR reaction with primer sets designed to amplify specific viral RNA regions is the method of choice to amplify human NoVs prior to downstream molecular analysis
Recent studies described the use of multiple genogroup I (GI) and genogroup II (GII)-specific degenerate primer sets for rRT-PCR to detect a wide range of GI and GII NoVs (Kageyama et al 2003; Kojima et al 2002; Richards et al 2004)
Summary
Noroviruses (NoVs) are recognized as the leading causative agents of outbreaks and sporadic cases of nonbacterial acute gastroenteritis across all ages in humans, resulting in more than 267,000,000 annual infections worldwide and over 200,000 deaths each year among children under 5 years old in developing countries (Noel et al 1999; Patel et al 2008; Donaldson et al 2008). The viruses are a broad range of enteric pathogens with great genetic and antigenic diversity (Wang et al 1994; Green et al 1995; Ando and Noel 2000). They segregate into 5 genogroups in which 3 genogroups (GI, GII, and GIV) are associated with human infection, with at least 8 genetic clusters in GI and 17 in GII (Zheng et al 2006). Reverse transcriptase polymerase chain reaction (RT-PCR) and subsequent genomic sequencing of the RT-PCR product have become the major means for
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
