Format

Send to:

Choose Destination
See comment in PubMed Commons below
J Microbiol Methods. 2012 Sep;90(3):228-34. doi: 10.1016/j.mimet.2012.05.012. Epub 2012 May 23.

Rapid detection and antimicrobial resistance gene profiling of Yersinia pestis using pyrosequencing technology.

Author information

  • 1Canadian Food Inspection Agency, National Centers for Animal Disease, Lethbridge Laboratory, Lethbridge, Alberta, Canada. kingsley.amoako@inspection.gc.ca

Abstract

When a bioterrorism attack is attempted or perpetrated there is considerable risk for public health and large scale socioeconomic consequences. It is imperative that we possess established assays for the rapid identification of biothreat agents with high sensitivity and specificity to ensure emergency response measures can be deployed appropriately. Highly trustworthy information within a relevant timeframe is required to make a rapid and informed decision. Obtaining DNA sequence data from a suspected agent provides an added layer of confidence compared to a presumptive positive PCR amplicon. Sequencing based technologies, such as pyrosequencing, have sufficient discrimination potential to be used for microbial identification and can also be used to identify antimicrobial resistance (AMR) genes. We have shown in this study the power of pyrosequencing in the unambiguous detection and identification of nine Yersinia pestis strains based on virulence genes. Furthermore, we developed assays to characterize their AMR gene profiles. Sequence results ranging from 40 to 84bp were generated in about 60 min following initial PCR amplification and provide a rapid method for determining the AMR profile as compared to the conventional plate method which takes several days. The high sequence identities (95-100%) and specificity observed indicate the high level of accuracy of pyrosequencing technology. In addition, the read lengths of up to 84 bp observed in this study are unprecedented for pyrosequencing using the Pyromark Q24. We propose this method as a novel, rapid, sequence based detection and identification tool for Y. pestis with a potential application in biodefence.

Crown Copyright © 2012. Published by Elsevier B.V. All rights reserved.

PMID:
22634001
[PubMed - indexed for MEDLINE]
PubMed Commons home

PubMed Commons

0 comments
How to join PubMed Commons

    Supplemental Content

    Full text links

    Icon for Elsevier Science
    Loading ...
    Write to the Help Desk