Abstract
Campylobacter spp. are important causes of bacterial gastroenteritis in humans in developed countries. Among Campylobacter spp. Campylobacter jejuni (C. jejuni) and C. coli are the most common causes of human infection. In this study, a multiplex PCR (mPCR) and high resolution melt (HRM) curve analysis were optimized for simultaneous detection and differentiation of C. jejuni and C. coli isolates. A segment of the hippuricase gene (hipO) of C. jejuni and putative aspartokinase (asp) gene of C. coli were amplified from 26 Campylobacter isolates and amplicons were subjected to HRM curve analysis. The mPCR-HRM was able to differentiate between C. jejuni and C. coli species. All DNA amplicons generated by mPCR were sequenced. Analysis of the nucleotide sequences from each isolate revealed that the HRM curves were correlated with the nucleotide sequences of the amplicons. Minor variation in melting point temperatures of C. coli or C. jejuni isolates was also observed and enabled some intraspecies differentiation between C. coli and/or C. jejuni isolates. The potential of PCR-HRM curve analysis for the detection and speciation of Campylobacter in additional human clinical specimens and chicken swab samples was also confirmed. The sensitivity and specificity of the test were found to be 100% and 92%, respectively. The results indicated that mPCR followed by HRM curve analysis provides a rapid (8 hours) technique for differentiation between C. jejuni and C. coli isolates.
Highlights
Thermophilic Campylobacters, C. jejuni and C. coli, are the leading causes of human foodborne bacterial gastroenteritis worldwide, and are of major public health significance [1].Campylobacter has been identified as the major source of food poisoning in the United States [2], Europe [1, 3] and Australia [4].PLOS ONE | DOI:10.1371/journal.pone.0138808 September 22, 2015multiplex Polymerase Chain Reaction (PCR) (mPCR)-high resolution melt (HRM) of Campylobacter spp.and analysis, decision to publish, or preparation of the manuscript
This study describes a rapid and reliable multiplex PCR (mPCR)-HRM technique for the differentiation of C. jejuni and C. coli isolates
C. jejuni and C. coli are very close in their phenotypic and genotypic characteristics, the present study shows that the mPCR-HRM technique was able to differentiate between C. jejuni and C. coli using hipO and asp genes, respectively
Summary
Thermophilic Campylobacters, C. jejuni and C. coli, are the leading causes of human foodborne bacterial gastroenteritis worldwide, and are of major public health significance [1].Campylobacter has been identified as the major source of food poisoning in the United States [2], Europe [1, 3] and Australia [4].PLOS ONE | DOI:10.1371/journal.pone.0138808 September 22, 2015mPCR-HRM of Campylobacter spp.and analysis, decision to publish, or preparation of the manuscript. Thermophilic Campylobacters, C. jejuni and C. coli, are the leading causes of human foodborne bacterial gastroenteritis worldwide, and are of major public health significance [1].Campylobacter has been identified as the major source of food poisoning in the United States [2], Europe [1, 3] and Australia [4]. Analysis, decision to publish, or preparation of the manuscript. The specific role of this author is articulated in the ‘author contributions’ section
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