title: Rapid detection and differentiation of human noroviruses using RT-PCR coupled to electrospray ionization mass spectrometry.
abstract: The goal of this study was to develop an assay for the detection and differentiation of noroviruses using RT-PCR followed by electrospray ionization mass spectrometry (ESI-MS). Detection of hepatitis A virus was also considered. Thirteen primer pairs were designed for use in this assay and a reference database was created using GenBank sequences and reference norovirus samples. The assay was tested for inclusivity and exclusivity using 160 clinical norovirus samples, 3 samples of hepatitis A virus and 3 other closely related viral strains. Results showed that the assay was able to detect norovirus with a sensitivity of 92% and a specificity of 100%. Norovirus identification at the genogroup level was correct for 98% of samples detected by the assay and for 75% of a subset of samples (n = 32) compared at the genotype level. Identification of norovirus genotypes is expected to improve as more reference samples are added to the database. The assay was also capable of detecting and genotyping hepatitis A virus in all 3 samples tested. Overall, the assay developed here allows for detection and differentiation of noroviruses within one working day and may be used as a tool in surveillance efforts or outbreak investigations.
authors.affiliation: Chapman University, Schmid College of Science and Technology, Food Science and Nutrition, One University Drive, Orange, CA 92866, USA. Electronic address: hellberg@chapman.edu.
authors.forename: Rosalee S
authors.initials: RS
authors.lastname: Hellberg
authors.affiliation: Ibis Biosciences, Abbott, 2251 Faraday Ave., Suite 150, Carlsbad, CA 92008, USA.
authors.forename: Feng
authors.initials: F
authors.lastname: Li
authors.affiliation: Ibis Biosciences, Abbott, 2251 Faraday Ave., Suite 150, Carlsbad, CA 92008, USA.
authors.forename: Rangarajan
authors.initials: R
authors.lastname: Sampath
authors.affiliation: Ibis Biosciences, Abbott, 2251 Faraday Ave., Suite 150, Carlsbad, CA 92008, USA.
authors.forename: Irene J
authors.initials: IJ
authors.lastname: Yasuda
authors.affiliation: Ibis Biosciences, Abbott, 2251 Faraday Ave., Suite 150, Carlsbad, CA 92008, USA.
authors.forename: Heather E
authors.initials: HE
authors.lastname: Carolan
authors.affiliation: Orange County Public Health Laboratory, 1729 West 17th Street, Santa Ana, CA 92706, USA.
authors.forename: Julia M
authors.initials: JM
authors.lastname: Wolfe
authors.affiliation: Orange County Public Health Laboratory, 1729 West 17th Street, Santa Ana, CA 92706, USA.
authors.forename: Michael K
authors.initials: MK
authors.lastname: Brown
authors.affiliation: Orange County Public Health Laboratory, 1729 West 17th Street, Santa Ana, CA 92706, USA.
authors.forename: Richard C
authors.initials: RC
authors.lastname: Alexander
authors.affiliation: U.S. Food and Drug Administration, Office of Regulatory Affairs, Pacific Regional Laboratory Southwest, 19701 Fairchild, Irvine, CA 92612, USA.
authors.forename: Donna M
authors.initials: DM
authors.lastname: Williams-Hill
authors.affiliation: U.S. Food and Drug Administration, Office of Regulatory Affairs, Pacific Regional Laboratory Southwest, 19701 Fairchild, Irvine, CA 92612, USA.
authors.forename: William B
authors.initials: WB
authors.lastname: Martin
mesh_terms: D017931:DNA Primers / Q000235:genetics; D005506:Food Contamination / Q000032:analysis*; D006801:Humans; D029322:Norovirus / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D017931:DNA Primers
keywords: Electrospray ionization mass spectrometry; Genotyping; Hepatitis A virus; Norovirus; RT-PCR