Mulitplex problems - (Apr/30/2007 )

Hi All

I am attempting to set up a multiplex for routine use in our lab to identify Campylobacter coli and jejuni using conventional pcr (not real-time). This method is for chicken rinses and also includes a 16s rDNA internal amplification control (IAC). All primers sets work individually at the same temperature and are specific for the species intended. When I run the multiplex, on pure cultures, it works perfectly and I get the expected fragmnets of either 344 bp (jejuni) or 500bp (coli) with the 16s amplification control, looks fantastic. However.........., when I add purified DNA from jejuni and coli to the same reaction tube, I only get the 344 bp fragment indicative of coli and the IAC (1062bp). There is not even any weak amplification at 500bp??????????? I would expect my future samples to have a both coli and jejuni, as it will be DNA purified from rinses and enrichment broths, and need to identify the presence of both, so this is a problem.

I am a lazy molecular biologist and use a commercially available mastermix, which means i cannot adjust my individual reagents in the mix. Could this be the source of my problem?? Would this still explain why i only get amplification when pure cultures are present? I am hesitant to order all the inidividual reagents as originally described in the paper unless I am confident that this is the source of the problem as I have no funding at present for this work

This method was described by Persson & Olsen (2005), Journal of Medical Microbiology, 54, 1043-1047. I noticed in this paper that they only work on pure cultures and when they do some work on stool samples they use camp negative stool samples and spike with either coli or jejuni, but not both.

Cheers
Tom

A common way to improve multiplex is to increase the concentration of buffer (mostly the KCl) by 1.5x. If you're already using commercially available mastermix... can you just add less water? Or is the entire mastermix made for you and you just add primers?

The next step is to increase the relative concentration of the 500bp primers. You might need to reduce the other primers to avoid "too-much-primer syndrome". Note that as a rule multiplex preferentially amplifies the smaller products over larger ones, so you almost always need to adjust the primers to compensate.

Thanks again for the tips. Increasing the amount of mastermix did not help, but adjusting the concentration of the primers did after several tweaks.

Many thanks
Tom