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P2-113 Development of a Novel Polymerase Chain Reaction Electrospray Ionization Mass Spectrometer (PCR/ESI-MS) Assay for the Detection and Differentiation of Human Noroviruses

Tuesday, July 24, 2012
Exhibit Hall (Rhode Island Convention Center)
Rosalee Hellberg, U.S. Food and Drug Administration, Irvine, CA
Feng Li, Ibis Biosciences, Carlsbad, CA
Rangarajan Sampath, Ibis Biosciences, Carlsbad, CA
Kyson Chou, U.S. Food and Drug Administration, Irvine, CA
Donna Williams-Hill, U.S. Food and Drug Administration, Irvine, CA
William Martin, U.S. FDA, Irvine, CA
Irene Yasuda, U.S. Food and Drug Administration, Ivine, CA
Heather Carolan, U.S. Food and Drug Administration, Irvine, CA
Introduction: Norovirus is the leading cause of foodborne illness in the United States; however, current methods for differentiation of genotypes and strains are time-consuming and inconsistent.  A novel method using PCR/ESI-MS may allow for detection and differentiation of noroviruses at the genotype and strain level within one working day.  This method uses the exact weight of a PCR amplicon to determine its base composition and then compares this measurement against a reference database to identify the sample. 

Purpose: The goal of this study was to develop a PCR/ESI-MS assay for the rapid detection and genetic differentiation of noroviruses.

Methods: To design primers for this assay, over 5,000 norovirus sequences were downloaded from GenBank.  Sequences were aligned with BioEdit and primers were designed to amplify all the human noroviruses.  Primers were analyzed in silico for parameters such as primer-dimer formation, % GC, and annealing temperature.  Predicted base compositions related to each genotype were determined and used to select the final primer sets that provided maximal differentiation amongst the various genotypes.

Results: Following the initial design and in silico analysis of primers, a total of 8 primer pairs were selected for use in the PCR/ESI-MS assay that are arranged in a 96-well plate format, allowing for analysis of 12 samples per plate.  These primer pairs amplify 50-150 bp fragments within the genes coding for the major capsid protein (VP1) and RNA-dependent RNA polymerase (RdRp).  Analysis of the amplicons with ESI-MS is predicted to enable differentiation of 30 human norovirus genotypes, as well as strains within the GII.4 genotype. 

Significance: The assay developed in this project will reduce the time and labor needed to detect and differentiate noroviruses and will enhance the ability of public health scientists to identify the source and the spread of norovirus illnesses related to food outbreak situations.

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