title: Differentiation of Campylobacter jejuni and Campylobacter coli Using Multiplex-PCR and High Resolution Melt Curve Analysis.
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.
authors.affiliation: School of Animal and Veterinary Sciences, Charles Sturt University, Wagga Wagga, NSW, Australia; Graham Centre for Agricultural Innovation (NSW Department of Primary Industries and Charles Sturt University), Wagga Wagga, NSW, Australia.
authors.forename: Banya
authors.initials: B
authors.lastname: Banowary
authors.affiliation: School of Animal and Veterinary Sciences, Charles Sturt University, Wagga Wagga, NSW, Australia; Graham Centre for Agricultural Innovation (NSW Department of Primary Industries and Charles Sturt University), Wagga Wagga, NSW, Australia.
authors.forename: Van Tuan
authors.initials: VT
authors.lastname: Dang
authors.affiliation: School of Animal and Veterinary Sciences, Charles Sturt University, Wagga Wagga, NSW, Australia; Graham Centre for Agricultural Innovation (NSW Department of Primary Industries and Charles Sturt University), Wagga Wagga, NSW, Australia.
authors.forename: Subir
authors.initials: S
authors.lastname: Sarker
authors.affiliation: School of Animal and Veterinary Sciences, Charles Sturt University, Wagga Wagga, NSW, Australia; Graham Centre for Agricultural Innovation (NSW Department of Primary Industries and Charles Sturt University), Wagga Wagga, NSW, Australia.
authors.forename: Joanne H
authors.initials: JH
authors.lastname: Connolly
authors.affiliation: Birling Avian Laboratories, Bringelly, NSW, Australia.
authors.forename: Jeremy
authors.initials: J
authors.lastname: Chenu
authors.affiliation: The University of Sydney, Sydney, NSW, Australia.
authors.forename: Peter
authors.initials: P
authors.lastname: Groves
authors.affiliation: School of Animal and Veterinary Sciences, Charles Sturt University, Wagga Wagga, NSW, Australia; Graham Centre for Agricultural Innovation (NSW Department of Primary Industries and Charles Sturt University), Wagga Wagga, NSW, Australia.
authors.forename: Michelle
authors.initials: M
authors.lastname: Ayton
authors.affiliation: School of Animal and Veterinary Sciences, Charles Sturt University, Wagga Wagga, NSW, Australia; Graham Centre for Agricultural Innovation (NSW Department of Primary Industries and Charles Sturt University), Wagga Wagga, NSW, Australia.
authors.forename: Shane
authors.initials: S
authors.lastname: Raidal
authors.affiliation: School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, NSW, Australia.
authors.forename: Aruna
authors.initials: A
authors.lastname: Devi
authors.affiliation: Graham Centre for Agricultural Innovation (NSW Department of Primary Industries and Charles Sturt University), Wagga Wagga, NSW, Australia; School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, NSW, Australia.
authors.forename: Thiru
authors.initials: T
authors.lastname: Vanniasinkam
authors.affiliation: School of Animal and Veterinary Sciences, Charles Sturt University, Wagga Wagga, NSW, Australia; Graham Centre for Agricultural Innovation (NSW Department of Primary Industries and Charles Sturt University), Wagga Wagga, NSW, Australia.
authors.forename: Seyed A
authors.initials: SA
authors.lastname: Ghorashi
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics; D002169:Campylobacter Infections / Q000382:microbiology; D017000:Campylobacter coli / Q000145:classification / Q000235:genetics*; D016123:Campylobacter jejuni / Q000145:classification / Q000235:genetics*; D058893:DNA Barcoding, Taxonomic / Q000379:methods*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics*; D014644:Genetic Variation; D005838:Genotype; D006801:Humans; D008969:Molecular Sequence Data; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D010802:Phylogeny; D017422:Sequence Analysis, DNA; D013045:Species Specificity; D044366:Transition Temperature
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial