I am looking for an internal control (IC) that can be spiked into clinical specimens pre-extraction devoid of any background E. coli gDNA. We currently use a plasmid containing a commercially synthesized gene, but keep picking up residual E. coli in our 16S profiles since the plasmid was transformed into E. coli. According to the manufacturer its close to impossible to completely remove all residual E. coli DNA from the plasmid prep process. Since E. coli is one of our targets, we are trying to move away from plasmid-based IC. We would also like to stay away from ssDNA viral IC such as MS2 because that would introduce an extra step (RT-PCR) in our workflow. Does anyone know of any commercially available or well established dsDNA viral/ bacteria/ eukaryotic IC we can adopt?
What is the purpose of your internal control? Simply to make sure the 16s amplification works? You could generate a PCR amplicon that includes internal binding sites for your primers; you wouldn't need to spike in very much at all.
The purpose of the IC is to ensure extraction and amplification were successful. I thought of that but I was worried using a long dsDNA or gene fragment would not provide any information on the extraction process, since they would not be packaged or encapsulated in anything that would require lysis.
I would suggest Agrobacterium tumifaciens. This is a plant pathogen used in plant genetic engineering, so some traces of its DNA might be found in persons who have eaten GM foods, but the only part of the A. tumifaciens genome that is transferred to the plants is a part of the Ti plasmid. Any other genes would make good IC targets and the bacterium has been extensively researched, so it shouldn't be difficult to find a good PCR in the literature; alternatively, you could just monitor the appearance of A. tumifaciens 16s RNA sequences in your NGS results.
A. tumifaciens is easy to grow in the lab, harmless to humans and, as far as I know, is never found in human samples - though traces may occur from environmental contamination.
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