Evaluation of multiplex-PCR and high-resolution melt curve analysis for differentiation of Campylobacter jejuni and Campylobacter coli

Seyed Ghorashi, Banya Banowary, Van Tuan Dang, Subir Sarker, Joanne Connolly, Peter Groves, Michelle Ayton, Shane Raidal, Thiru Vanniasinkam

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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.
Key words
Campylobacter, multiplex-PCR, high resolution melt curve analysis
Original languageEnglish
Publication statusPublished - 15 Nov 2017
EventAustralian Veterinary Poultry Association Scientific Meeting - Australia, Geelong, Australia
Duration: 15 Nov 201717 Nov 2017
http://www.avpa.asn.au/avpa-scientific-meeting-geelong-15th-17th-november-2017/

Conference

ConferenceAustralian Veterinary Poultry Association Scientific Meeting
CountryAustralia
CityGeelong
Period15/11/1717/11/17
Internet address

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    Ghorashi, S., Banowary, B., Dang, V. T., Sarker, S., Connolly, J., Groves, P., Ayton, M., Raidal, S., & Vanniasinkam, T. (2017). Evaluation of multiplex-PCR and high-resolution melt curve analysis for differentiation of Campylobacter jejuni and Campylobacter coli. Paper presented at Australian Veterinary Poultry Association Scientific Meeting, Geelong, Australia.