Veterinary Laboratories Agency (Weybridge), New Haw, Addlestone, Surrey KT15 3NB, UK.
J Med Microbiol. 2010 Feb;59(Pt 2):172-178. doi: 10.1099/jmm.0.014415-0. Epub 2009 Oct 15.
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.
常规和实时 PCR 检测的一个常见问题是由于样品中存在抑制物质而导致 DNA 扩增失败。有鉴于此，我们的目的是开发和评估用于现有的大肠弯曲杆菌和空肠弯曲杆菌双重实时 PCR 检测的内部扩增对照 (IAC)。我们开发并评估了竞争性和非竞争性 IAC。竞争性方法涉及编码区的 DNA 片段鱼病毒性出血性败血症病毒，侧翼由 mapA PCR 引物，而非竞争性方法则利用了一套额外的通用 16S rDNA 引物。使用弯曲杆菌培养物和鸡盲肠内容物样本评估了这两种 IAC-PCR 检测类型。两种 IAC 对盲肠抑制剂均敏感，使其适合检测可能导致假阴性的抑制作用。结果表明，两种 IAC 在最佳浓度下均能很好地工作，而不会降低 PCR 检测的整体敏感性。与培养物相比，优化的竞争性 IAC-PCR 检测法检测到 47 个阳性中的 45 个（灵敏度 93.6%，特异性 80.1%）；然而，它相对于培养物具有优势，因为它可以检测大肠弯曲杆菌和空肠弯曲杆菌的混合感染，并且能够在一天内为样本提供结果。
