Lund M, Madsen M
Danish Institute for Food and Veterinary Research (DFVF), Arhus N, Denmark.
Mol Cell Probes. 2006 Apr;20(2):92-9. doi: 10.1016/j.mcp.2005.10.002. Epub 2005 Dec 5.
To illustrate important issues in optimization of a PCR assay with an internal control four different primer combinations for conventional PCR, two non-competitive and two competitive set-ups for real time PCR were used for detection of Campylobacter spp. in chicken faecal samples. In the conventional PCR assays the internal control was genomic DNA from Yersinia ruckeri, which is not found in chicken faeces. This internal control was also used in one of the set ups in real time PCR. In the three other set-ups different DNA fragments of 109 bp length prepared from two oligos of each 66 bp by a simple extension reaction was used. All assays were optimized to avoid loss of target sensitivity due to the presence of the internal control by adjusting the amount of internal control primers in the duplex assays and the amount of internal control in all assays. Furthermore, the assays were tested against faecal inhibitors to ensure that the internal control and the target PCR had the same sensitivity towards inhibitors.
为了阐述在使用内部对照优化聚合酶链反应（PCR）检测方法时的重要问题，采用了四种不同的常规PCR引物组合、两种非竞争性和两种竞争性实时PCR设置来检测鸡粪便样本中的弯曲杆菌属。在常规PCR检测中，内部对照是来自鲁氏耶尔森菌的基因组DNA，鸡粪便中不存在这种菌。该内部对照也用于其中一种实时PCR设置。在其他三种设置中，通过简单的延伸反应，使用了由两个66 bp的寡核苷酸制备的长度为109 bp的不同DNA片段。通过在双重检测中调整内部对照引物的量以及在所有检测中调整内部对照的量，对所有检测进行了优化，以避免由于内部对照的存在而导致目标灵敏度的损失。此外，针对粪便抑制剂对检测进行了测试，以确保内部对照和目标PCR对抑制剂具有相同的灵敏度。
