title: A norovirus detection architecture based on isothermal amplification and expanded genetic systems.
abstract: Noroviruses are the major cause of global viral gastroenteritis with short incubation times and small inoculums required for infection. This creates a need for a rapid molecular test for norovirus for early diagnosis, in the hope of preventing the spread of the disease. Non-chemists generally use off-the shelf reagents and natural DNA to create such tests, suffering from background noise that comes from adventitious DNA and RNA (collectively xNA) that is abundant in real biological samples, especially feces, a common location for norovirus. Here, we create an assay that combines artificially expanded genetic information systems (AEGIS, which adds nucleotides to the four in standard xNA, pairing orthogonally to A:T and G:C) with loop-mediated isothermal amplification (LAMP) to amplify norovirus RNA at constant temperatures, without the power or instrument requirements of PCR cycling. This assay was then validated using feces contaminated with murine norovirus (MNV). Treating stool samples with ammonia extracts the MNV RNA, which is then amplified in an AEGIS-RT-LAMP where AEGIS segments are incorporated both into an internal LAMP primer and into a molecular beacon stem, the second lowering background signaling noise. This is coupled with RNase H nicking during sample amplification, allowing detection of as few as 10 copies of noroviral RNA in a stool sample, generating a fluorescent signal visible to human eye, all in a closed reaction vessel.
authors.affiliation: Foundation for Applied Molecular Evolution (FfAME), 13709 Progress Boulevard, # 7, Alachua, FL 32615, USA.
authors.forename: Ozlem
authors.initials: O
authors.lastname: Yaren
authors.affiliation: Foundation for Applied Molecular Evolution (FfAME), 13709 Progress Boulevard, # 7, Alachua, FL 32615, USA.
authors.forename: Kevin M
authors.initials: KM
authors.lastname: Bradley
authors.affiliation: Firebird Biomolecular Sciences LLC, 13709 Progress Boulevard, # 17, Alachua, FL 32615, USA.
authors.forename: Patricia
authors.initials: P
authors.lastname: Moussatche
authors.affiliation: Foundation for Applied Molecular Evolution (FfAME), 13709 Progress Boulevard, # 7, Alachua, FL 32615, USA.
authors.forename: Shuichi
authors.initials: S
authors.lastname: Hoshika
authors.affiliation: Foundation for Applied Molecular Evolution (FfAME), 13709 Progress Boulevard, # 7, Alachua, FL 32615, USA.
authors.forename: Zunyi
authors.initials: Z
authors.lastname: Yang
authors.affiliation: Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, Gainesville FL 32611, USA; The Emerging Pathogens Institute, University of Florida, Gainesville FL 32611, USA.
authors.forename: Shu
authors.initials: S
authors.lastname: Zhu
authors.affiliation: Department of Molecular Genetics and Microbiology, College of Medicine, University of Florida, Gainesville FL 32611, USA; The Emerging Pathogens Institute, University of Florida, Gainesville FL 32611, USA.
authors.forename: Stephanie M
authors.initials: SM
authors.lastname: Karst
authors.affiliation: Firebird Biomolecular Sciences LLC, 13709 Progress Boulevard, # 17, Alachua, FL 32615, USA. Electronic address: manuscripts@ffame.org.
authors.forename: Steven A
authors.initials: SA
authors.lastname: Benner
mesh_terms: D017250:Caliciviridae Infections / Q000175:diagnosis* / Q000821:virology; D017931:DNA Primers / Q000235:genetics; D005243:Feces / Q000821:virology*; D005759:Gastroenteritis / Q000175:diagnosis* / Q000821:virology; D006801:Humans; D015335:Molecular Probes; D029322:Norovirus / Q000235:genetics* / Q000302:isolation & purification*; D021141:Nucleic Acid Amplification Techniques / Q000379:methods*; D012367:RNA, Viral / Q000235:genetics / Q000302:isolation & purification*; D016914:Ribonuclease H / Q000235:genetics / Q000378:metabolism; D012680:Sensitivity and Specificity; D013696:Temperature
chemical_list: D017931:DNA Primers; D015335:Molecular Probes; D012367:RNA, Viral; D016914:Ribonuclease H
keywords: AEGIS; Norovirus; RNase H nicking molecular beacon; RT-LAMP