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Detection of norovirus capsid protein in authentic standards and in stool extracts by matrix-assisted laser desorption ionization and nanospray mass spectrometry.

Authors
Colquhoun-DR; Schwab-KJ; Cole-RN; Halden-RU
Source
Appl Environ Microbiol 2006 Apr; 72(4):2749-2755
NIOSHTIC No.
20038257
Abstract
Mass spectrometry (MS) represents a rapid technique for the identification of microbial monocultures, and its adaptation to the detection of pathogens in real-world samples is a public health and homeland security priority. Norovirus, a leading cause of gastroenteritis in the world, is difficult to monitor because it cannot be cultured outside the human body. The detection of norovirus capsid protein was explored using three common MS-based methods: scanning of intact proteins, peptide mass fingerprinting, and peptide sequencing. Detection of intact target protein was limited by poor selectivity and sensitivity. Detection of up to 16 target peptides by peptide mass fingerprinting allowed for the reproducible and confident (P < 0.05) detection of the 56-kDa norovirus capsid protein in the range of 0.1 x 10(-12) to 50 x 10(-12) mol in authentic standards of recombinant norovirus virus-like particles (VLPs). To explore assay performance in complex matrixes, a non-gel-based, rapid method (2 to 3 h) for virus extraction from human stool was evaluated (72% +/- 12% recovery), and additional analyses were performed on norovirus-free stool extracts fortified with VLPs. Whereas peptide mass fingerprinting was rendered impractical by sample interferences, peptide sequencing using nanospray tandem MS facilitated unambiguous identification of > or =250 fmol of capsid protein in stool extracts. This is the first report on MS-based detection of norovirus, accomplished by using structurally identical, noninfective VLPs at clinically relevant concentrations. It represents an important milestone in the development of assays for surveillance of this category B bioterrorism agent.
Keywords
Amino-acids; Gastrointestinal-system; Gastrointestinal-system-disorders; Humans; Viral-diseases; Viral-infections
Contact
Rolf U. Halden, JHU Center for Water and Health, Department of Environmental Health Sciences, Bloomberg School of Public Health, Johns Hopkins University, BSPH Bldg., Room E6618, Baltimore, MD 21205
CODEN
AEMIDF
Publication Date
20060401
Document Type
Journal Article
Email Address
rhalden@jhsph.edu
Funding Type
Grant
Fiscal Year
2006
NTIS Accession No.
NTIS Price
Identifying No.
Grant-Number-T42-OH-008428
Issue of Publication
4
ISSN
0099-2240
Source Name
Applied and Environmental Microbiology
State
MD
Performing Organization
Johns Hopkins University
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