title: Development and evaluation of internal amplification controls for use in a real-time duplex PCR assay for detection of Campylobacter coli and Campylobacter jejuni.
abstract: A common problem of both conventional and real-time PCR assays is failure of DNA amplification due to the presence of inhibitory substances in samples. In view of this, our aim was to develop and evaluate internal amplification controls (IACs) for use with an existing duplex real-time PCR assay for Campylobacter coli and Campylobacter jejuni. Both competitive and non-competitive IACs were developed and evaluated. The competitive approach involved a DNA fragment of the coding region of the fish viral haemorrhagic septicaemia virus, flanked by the mapA PCR primers, whilst the non-competitive approach utilized an extra set of universal 16S rDNA primers. Both IAC-PCR assay types were evaluated using cultures of Campylobacter and chicken caecal content samples. Both IACs were sensitive to caecal inhibitors, making them suitable for detecting inhibition which could lead to false-negatives. Results showed that both IACs at optimum concentrations worked well without reducing the overall sensitivity of the PCR assay. Compared to culture, the optimized competitive IAC-PCR assay detected 45/47 positives (sensitivity 93.6 %, specificity 80.1 %); however, it had the advantage over culture in that it could detect mixed infections of C. coli and C. jejuni and was capable of giving a result for a sample within a day.
authors.affiliation: Veterinary Laboratories Agency (Weybridge), New Haw, Addlestone, Surrey KT15 3NB, UK.
authors.forename: Luke
authors.initials: L
authors.lastname: Randall
authors.affiliation: Veterinary Laboratories Agency (Weybridge), New Haw, Addlestone, Surrey KT15 3NB, UK.
authors.forename: Fabrizio
authors.initials: F
authors.lastname: Lemma
authors.affiliation: Veterinary Laboratories Agency (Weybridge), New Haw, Addlestone, Surrey KT15 3NB, UK.
authors.forename: John
authors.initials: J
authors.lastname: Rodgers
authors.affiliation: Veterinary Laboratories Agency (Weybridge), New Haw, Addlestone, Surrey KT15 3NB, UK.
authors.forename: Ana
authors.initials: A
authors.lastname: Vidal
authors.affiliation: Veterinary Laboratories Agency (Weybridge), New Haw, Addlestone, Surrey KT15 3NB, UK.
authors.forename: Felicity
authors.initials: F
authors.lastname: Clifton-Hadley
mesh_terms: D000818:Animals; D001431:Bacteriological Techniques; D002169:Campylobacter Infections / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D017000:Campylobacter coli / Q000302:isolation & purification*; D016123:Campylobacter jejuni / Q000302:isolation & purification*; D002432:Cecum / Q000382:microbiology; D002645:Chickens / Q000382:microbiology; D005766:Gastrointestinal Contents / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D011201:Poultry Diseases / Q000175:diagnosis / Q000382:microbiology; D012680:Sensitivity and Specificity; D013045:Species Specificity