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Development and Evaluation of Internal Amplification Controls (IACs) for use in a real time duplex PCR assay for detection of Campylobacter coli and C. jejuni

VLA Weybridge

¹ E-mail: l.randall{at}vla.defra.gsi.gov.uk

Received July 16, 2009
Accepted October 8, 2009

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 C. jejuni. Both competitive and non-competitive IACs were developed and evaluated. The competitive approach involved a DNA fragment of the coding region of the viral fish haemorrhagic septicaemia virus, flanked by the mapA PCR primers, whilst the non-competitive approach utilised an extra set of universal 16S 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 to detect 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. 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.