Poster Presentation Australian Society for Microbiology Annual Scientific Meeting 2014

Genotyping of Campylobacter jejuni and Campylobacter coli using multiplex-PCR and high resolution melt curve analysis (#249)

Banya Banowary 1 2 , Subir Sarker 1 2 , Joanne J Connolly 1 2 , Jeremy Chenu 3 , Peter Groves 4 , Michelle Ayton 1 2 , Shane Raidal 1 2 , Seyed Ali Ghorashi 1 2
  1. Department of Primary Industries , Graham Centre for Agricultural Innovation, Wagga Wagga, NSW, Australia
  2. Charles Sturt University, Wagga Wagga, NSW, Australia
  3. Birling Avian Laboratories, Bringelly, NSW, Australia
  4. The University of Sydney, Sydney, NSW, Australia
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 was optimised 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 24 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 highly 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 results indicated that mPCR followed by HRM curve analysis provides a rapid (8 hours) and robust technique for differentiation between C. jejuni and C. coli isolates.