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United States Department of Agriculture

Agricultural Research Service

Research Project: MOLECULAR BIOLOGY AND GENOMICS OF FOODBORNE PATHOGENS

Location: Produce Safety and Microbiology Research

Title: High Resolution Genotyping of Campylobacter Using PCR and High-Throughput Mass Spectrometry

Authors
item Hannis, James - IBIS BIOSCIENCES
item Manalili, Sheri - IBIS BIOSCIENCES
item Hall, Thomas - IBIS BIOSCIENCES
item Ranken, Raymond - IBIS BIOSCIENCES
item White, N. - IBIS BIOSCIENCES
item Sampath, Rangarajan - IBIS BIOSCIENCES
item Blyn, Lawrence - IBIS BIOSCIENCES
item Ecker, David - IBIS BIOSCIENCES
item Mandrell, Robert
item Fagerquist, Clifton
item Bates, Anne
item Miller, William
item Hofstadler, Steven - IBIS BIOSCIENCES

Submitted to: Journal of Clinical Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: February 5, 2008
Publication Date: April 2, 2008
Citation: Hannis, J., Manalili, S., Hall, T., Ranken, R., White, N., Sampath, R., Blyn, L., Ecker, D., Mandrell, R.E., Fagerquist, C.K., Bates, A.H., Miller, W.G., Hofstadler, S. 2008. High Resolution Genotyping of Campylobacter Using PCR and High-Throughput Mass Spectrometry. Journal of Clinical Microbiology.46(4) 1220-1225.

Interpretive Summary: In this work we report a high throughput mass spectrometry-based technique for rapid high resolution strain identification of Campylobacter jejuni. This method readily distinguishes C. jejuni from C. coli, has comparable resolving power to multi-locus sequence typing (MLST), is applicable to mixtures, and is highly automated. The strain typing approach is based on high performance mass spectrometry which “weighs” PCR amplicons with enough mass accuracy to unambiguously determine the base composition of each amplicon (i.e., the number of A’s, G’s, C’s and T’s). Amplicons are derived from PCR primers which amplify short (< 140 bp) regions of housekeeping genes used by conventional MLST strategies. Results are presented from a challenge panel comprised of 25 strain types of C. jejuni and 25 strain types of C. Coli. These samples were parsed and resolved with demonstrated sensitivity down to 10 genomes/PCR reaction from pure isolates.

Technical Abstract: In this work we report a high throughput mass spectrometry-based technique for rapid high resolution strain identification of Campylobacter jejuni. This method readily distinguishes C. jejuni from C. coli, has comparable resolving power to multi-locus sequence typing (MLST), is applicable to mixtures, and is highly automated. The strain typing approach is based on high performance mass spectrometry which “weighs” PCR amplicons with enough mass accuracy to unambiguously determine the base composition of each amplicon (i.e., the number of A’s, G’s, C’s and T’s). Amplicons are derived from PCR primers which amplify short (< 140 bp) regions of housekeeping genes used by conventional MLST strategies. Results are presented from a challenge panel comprised of 25 strain types of C. jejuni and 25 strain types of C. Coli. These samples were parsed and resolved with demonstrated sensitivity down to 10 genomes/PCR reaction from pure isolates.

Last Modified: 4/18/2014
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