Document Detail


Use of clustered regularly interspaced short palindromic repeat sequence polymorphisms for specific detection of enterohemorrhagic Escherichia coli strains of serotypes O26:H11, O45:H2, O103:H2, O111:H8, O121:H19, O145:H28, and O157:H7 by real-time PCR.
MedLine Citation:
PMID:  23035199     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
We explored the genetic diversity of the clustered regularly interspaced short palindromic repeat (CRISPR) regions of enterohemorrhagic Escherichia coli (EHEC) to design simplex real-time PCR assays for each of the seven most important EHEC serotypes worldwide. A panel of 958 E. coli strains investigated for their CRISPR loci by high-throughput real-time PCR showed that CRISPR polymorphisms in E. coli strongly correlated with both O:H serotypes and the presence of EHEC virulence factors (stx and eae genes). The CRISPR sequences chosen for simplex real-time PCR amplification of EHEC strains belonging to the top 7 EHEC serogroups differentiated clearly between EHEC and non-EHEC strains. Specificity estimates for the CRISPR PCR assays varied from 97.5% to 100%. Sensitivity estimates for the assays ranged from 95.7% to 100%. The assays targeting EHEC O145:H28, O103:H2, and O45:H2 displayed 100% sensitivity. The combined usage of two simplex PCR assays targeting different sequences of the O26 CRISPR locus allowed detection of EHEC O26:H11 with 100% sensitivity. By combining two simplex PCR assays targeting different sequences of the EHEC O157 CRISPR locus, EHEC O157:H7 was detected with 99.56% sensitivity. EHEC O111:H8 and EHEC O121:H19 were detected with 95.9% and 95.7% sensitivity, respectively. This study demonstrates that the identification of EHEC serotype-specific CRISPR sequences is more specific than the mere identification of O-antigen gene sequences, as is used in current PCR protocols for detection of EHEC strains.
Authors:
Sabine Delannoy; Lothar Beutin; Patrick Fach
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Publication Detail:
Type:  Evaluation Studies; Journal Article     Date:  2012-10-03
Journal Detail:
Title:  Journal of clinical microbiology     Volume:  50     ISSN:  1098-660X     ISO Abbreviation:  J. Clin. Microbiol.     Publication Date:  2012 Dec 
Date Detail:
Created Date:  2012-11-19     Completed Date:  2013-04-17     Revised Date:  2013-07-11    
Medline Journal Info:
Nlm Unique ID:  7505564     Medline TA:  J Clin Microbiol     Country:  United States    
Other Details:
Languages:  eng     Pagination:  4035-40     Citation Subset:  IM    
Affiliation:
Anses (French Agency for Food, Environmental and Occupational Health and Safety), Food Safety Laboratory, Maisons-Alfort, France.
Data Bank Information
Bank Name/Acc. No.:
GENBANK/JX219753;  JX219754;  JX219755;  JX219756;  JX219757;  JX219758;  JX219759;  JX219760;  JX402083
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MeSH Terms
Descriptor/Qualifier:
Animals
Bacteriological Techniques / methods*
DNA, Bacterial / chemistry,  genetics*
Enterohemorrhagic Escherichia coli / classification*,  genetics,  isolation & purification*
Escherichia coli Infections / microbiology*
Genetic Variation
Humans
Inverted Repeat Sequences*
Molecular Sequence Data
Real-Time Polymerase Chain Reaction / methods*
Sensitivity and Specificity
Sequence Analysis, DNA
Chemical
Reg. No./Substance:
0/DNA, Bacterial
Comments/Corrections

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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