Campylobacter jejuni qPCR targeting VS1 gene - (Feb/25/2015 )

Hi there,

We are using a qPCR that is targeted to the VS1 gene (X71603.1) of C.jejuni that apparently is specific to C. jejuni and exists in a ratio of 1 copy per cell (Yang et al. 2003, Stonnet and Guesdon 1993). We are targeting the same VS1 gene as in Yang, but using different primers and probes due to our differences in qPCR platforms (they use a Roche cycler, and we have a ViiA7).

Although our laboratory strain of C. jejuni, ATCC 33291, seems to exist in this 1:1 ratio, it appears as if some clinical strains of C. jejuni (obtained from patient stool samples) do not. We have made standard curves with the laboratory strain as well as many clinical samples, and have found that with the lab strain the first standard of 10^7 copies of VS1 amplifies at around 20 Ct's. However, some of the Clinical samples at this quantity are amplifying much earlier at 13 Ct's and some much later at around 30 Ct's. This trend follows for the rest of the curve, down to 10^0. Since all of our standards have been calculated and prepared to have the exact same concentration regardless of which strain is used based on a 1:1 gene to cell ratio and a genome size of approx 1.7 million bp, this suggests there might be some variation in the number of gene copies for VS1 per cell in these clinical strains.

This result has been replicated numerous times. We always use fresh master mix, and have also re-extracted our DNA via a Qiagen kit.

We have done extensive BLAST searches and literature review, but all we seem to come up with is that this VS1 gene is specific to C. jejuni exclusively and that it does exist in a 1:1 ratio. We are quite confident in the manner we have quantified our DNA extractions as well.

I have attached a sample amplification plot to illustrate this result.

Any help or insight would be greatly appreciated,

Thanks,

Sophie

-Sophie1320-

You could try a quantitative next-gen sequencing approach to the samples that appear to have more or less than one copy and see if it is a real phenomenon. It would probably also pay to check the genome size of those ones, just to make sure you don't have either a contaminant or a genome duplication (or something similar).

-bob1-

Also, you could use inverse PCR to amplify the region surrounding the VS1 gene in your genomes. If there is truly several copies, there will be variation in the length of the PCR products. You could gel isolate to determine the flanking DNA.

-phage434-