title: Sequence-independent amplification coupled with DNA microarray analysis for detection and genotyping of noroviruses.
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.
authors.affiliation: Northeast Region Laboratory, Office of Regulatory Affairs, Food and Drug Administration, Jamaica, NY, USA. yuan.hu@fda.hhs.gov.
authors.forename: Yuan
authors.initials: Y
authors.lastname: Hu
authors.affiliation: Department of Microbiology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou, China. yanhi@mail.sysu.edu.cn.
authors.forename: Huijun
authors.initials: H
authors.lastname: Yan
authors.affiliation: Division of Molecular Biology, Center for Food Safety and Applied Nutrition, Food and Drug Administration, 8301 Muirkirk Road, Laurel, MD, 20708, USA. mark.mammel@fda.hhs.gov.
authors.forename: Mark
authors.initials: M
authors.lastname: Mammel
authors.affiliation: Division of Molecular Biology, Center for Food Safety and Applied Nutrition, Food and Drug Administration, 8301 Muirkirk Road, Laurel, MD, 20708, USA. haifeng.chen@fda.hhs.gov.
authors.forename: Haifeng
authors.initials: H
authors.lastname: Chen
keywords: DNA microarray; Detection; Genotyping; Noroviruses; Sequence-independent amplification