High-resolution melting analysis for rapid detection of sequence type 131 Escherichia coli

Lucas B. Harrison, Nancy D. Hanson

Research output: Contribution to journalArticle

2 Citations (Scopus)

Abstract

Escherichia coli isolates belonging to the sequence type 131 (ST131) clonal complex have been associated with the global distribution of fluoroquinolone and-lactam resistance. Whole-genome sequencing and multilocus sequence typing identify sequence type but are expensive when evaluating large numbers of samples. This study was designed to develop a cost-effective screening tool using highresolution melting (HRM) analysis to differentiate ST131 from non-ST131 E. coli in large sample populations in the absence of sequence analysis. The method was optimized using DNA from 12 E. coli isolates. Singleplex PCR was performed using 10 ng of DNA, Type-it HRM buffer, and multilocus sequence typing primers and was followed by multiplex PCR. The amplicon sizes ranged from 630 to 737 bp. Melt temperature peaks were determined by performing HRM analysis at 0.1°C resolution from 50 to 95°C on a Rotor-Gene Q 5-plex HRM system. Derivative melt curves were compared between sequence types and analyzed by principal component analysis. A blinded study of 191 E. coli isolates of ST131 and unknown sequence types validated this methodology. This methodology returned 99.2% specificity (124 true negatives and 1 false positive) and 100% sensitivity (66 true positives and 0 false negatives). This HRM methodology distinguishes ST131 from non-ST131 E. coli without sequence analysis. The analysis can be accomplished in about 3 h in any laboratory with an HRMcapable instrument and principal component analysis software. Therefore, this assay is a fast and cost-effective alternative to sequencing-based ST131 identification.

Original languageEnglish (US)
Article numbere00265-17
JournalAntimicrobial Agents and Chemotherapy
Volume61
Issue number6
DOIs
StatePublished - Jun 1 2017

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Freezing
Escherichia coli
Multilocus Sequence Typing
Principal Component Analysis
Sequence Analysis
vif Genes
Costs and Cost Analysis
Lactams
Multiplex Polymerase Chain Reaction
DNA
Fluoroquinolones
Buffers
Software
Genome
Polymerase Chain Reaction
Temperature
Population

All Science Journal Classification (ASJC) codes

  • Pharmacology
  • Pharmacology (medical)
  • Infectious Diseases

Cite this

High-resolution melting analysis for rapid detection of sequence type 131 Escherichia coli. / Harrison, Lucas B.; Hanson, Nancy D.

In: Antimicrobial Agents and Chemotherapy, Vol. 61, No. 6, e00265-17, 01.06.2017.

Research output: Contribution to journalArticle

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