What methods of extraction have you already tried? You would probably have more luck with a commercial method, such as QIAgen's suite of DNA extraction kits or MoBio's PowerSoil kit. Another approach, if you are looking for bacterial or eukaryotic DNA would be to spin down the urine for e.g. 5 min at 8000 rpm (not too hard) in a microcentrifuge, discard the supernatant, and continue the DNA extraction with the pellet. You could also wash that pellet with some PBS and spin down again. That should also get rid of many soluble inhibitors. Good luck!

Dear Elisabth;

Thank you so much for valuable comment. really I try to extracte the Toxoplasma gondii - a protozoan parasite- DNA with a low number in human urine for running a PCR but there are some problems due to high amount of urea and salt probably. I would appreciate your comments.

regards

Abba s Ali Eskandarian

Try using Recombinase Polymerase Amplification instead of PCR. It can tolerate a lot more urea as it's isothermal and works at ~37C. You get the same sensitivity as PCR, but reactions take 10-15 minutes. Check out http://www.twistdx.co.uk/our_technology/

Dear Matthew

Thank you. please send me some more techniqual detaile about your technology.

Should I design the primer and send you theire sequences?

As Elisabeth Bik mentioned, pelleting the material by centrifugation would be a good start. If you resuspend your pellet in a urea-free buffer such as PBS and re-centrifuge, your sample should be urea-free.

Alternatively, you could consider dialysis. Some manufacturers offer microdialysis apparatus (try Millipore or Pierce) which allows you to dialyse volumes in the 50 - 100 microliter range. Urea is a small, very soluble molecule, so a few hours' dialysis should be quite sufficient.

I do not know much about the presence of Toxoplasma in urine; are whole parasites/eggs present in urine, or only DNA? The centrifugation step would only make sense if the eggs/parasites are present, not if only DNA is present, since it would be in solution and not pellet.

If only DNA is present, I found this older paper that might provide a low-cost, kit-independent solution: http://www.ncbi.nlm.nih.gov/pubmed/9300381

Good luck!

HI

Thanks a lot to all. I try more with considering your comments.

A Eskandarian

Dear Abbas,

The original RPA publication is:

http://www.ncbi.nlm.nih.gov/pubmed/16756388

We have improved the technology since then to make it faster, but the basic premises are the same.

Information on how to pick primers and probes is in the manual and appendix:

http://www.twistdx.co.uk/images/uploads/docs/Combined_Manual-12-1.pdf

http://www.twistdx.co.uk/images/uploads/docs/Appendix.pdf

It might also be worth looking at the FAQs:

http://www.twistdx.co.uk/resources/faqs/

As you are hoping to perform a real-time assay, I would recommend using a TwistAmp Exo kit. The quick guide to this is here, so you can get an idea of the workflow:

http://www.twistdx.co.uk/images/uploads/docs/Quickguide_-_TwistAmp™_exo.pdf

We sell freeze dried reactions, it is up to end users to provide primers and probes of they wish to use them. The reactions contain all the dNTPs, enzymes etc, you just need to provide oligos and samples. There are a few external publications now, that you can find at:

http://www.twistdx.co.uk/resources/publications/

Best wishes,

Matthew

Dear Natthew

Thank you. I will read it carefully.

If you are looking for Toxoplasma gondii from urine without any carryover contamination of urea you should check either Urine DNA Isolation Kit for Exfoliated Cells or Bacteria or Bacterial Genomic DNA Isolation Kit from Norgen Biotek. They have optimized the isolation of DNA (being human, viral or Bacterial) from urine where the isolated DNA is inhibitor/urea-free and will not interfere with your PCR. Also they are also selling an optimized primers and positive control for Toxoplasma gondii. Good Luck

Try the new PureLink Microbiome DNA Purification Kit – it has good protocols for urine, stool and other samples http://www.thermofisher.com/order/catalog/product/A29790?ICID=search-product