title: Multiplex real-time RT-PCR for the simultaneous detection and quantification of GI, GII and GIV noroviruses.
abstract: Noroviruses are important causes of acute gastroenteritis and are classified into six genogroups with GI, GII and GIV containing human pathogens. This high genetic diversity represents a significant challenge for diagnostic assay development. Genogroup specific monoplex and multiplex real time RT-PCR assays are widely used for the detection of GI and GII noroviruses. On the other hand, GIV norovirus detection is not part of routine laboratory diagnosis. This study describes the development and evaluation of a one tube, real time RT-PCR assay for the simultaneous detection and quantification of GI, GII and GIV noroviruses, including both GIV.1 (human) and GIV.2 (animal) strains. Assay performance was evaluated on a panel of norovirus positive clinical samples by comparison of monoplex and multiplex standard curves and Ct values. The multiplex assay demonstrated equal sensitivity and specificity to the monoplex assays and was able to detect all GI, GII and GIV noroviruses with Ct values equal to that of the monoplex assays. The multiplex assay described in this study will be instrumental for the better understanding of GIV norovirus epidemiology, including their possible zoonotic nature.
authors.affiliation: Laboratory of Specialized Clinical Studies, Division of Infectious Diseases, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, United States. Electronic address: tibor.farkas@cchmc.org.
authors.forename: Tibor
authors.initials: T
authors.lastname: Farkas
authors.affiliation: Laboratory of Specialized Clinical Studies, Division of Infectious Diseases, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, United States.
authors.forename: Amy
authors.initials: A
authors.lastname: Singh
authors.affiliation: Laboratoire de Microbiologie IFREMER, Nantes, France.
authors.forename: Françoise S
authors.initials: FS
authors.lastname: Le Guyader
authors.affiliation: Istituto Superiore di Sanità, Department of Environment and Primary Prevention, Rome, Italy.
authors.forename: Giuseppina
authors.initials: G
authors.lastname: La Rosa
authors.affiliation: Food Animal Health Research Program, Ohio Agricultural Research and Development Center, College of Food, Agricultural and Environmental Sciences, Department of Veterinary Preventive Medicine, The Ohio State University, Wooster, OH, United States.
authors.forename: Linda
authors.initials: L
authors.lastname: Saif
authors.affiliation: Laboratory of Specialized Clinical Studies, Division of Infectious Diseases, Cincinnati Children's Hospital Medical Center, Cincinnati, OH, United States.
authors.forename: Monica
authors.initials: M
authors.lastname: McNeal
mesh_terms: D000818:Animals; D017250:Caliciviridae Infections / Q000175:diagnosis* / Q000662:veterinary / Q000821:virology*; D005838:Genotype; D006801:Humans; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D029322:Norovirus / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D019562:Viral Load / Q000379:methods
keywords: Calicivirus; Diagnosis; Norovirus; Real time RT-PCR