I've extracted gDNA from horse Urine for DNA Profiling according IQ System protocol (Promega). Unfortunately electropherogram showed high percentage of non-specific peak making impossible DNA Profiling. This is likely due to bacterial DNA interfering with the multiplex-PCR (I guess). I can say that cause a white pellet was obtained during the centrifugation step (before to proceed with the extraction protocol). Does anyone know how to avoid/remove Bacterial DNA while extracting animal DNA in Urine samples?
Thanks
Your question reminded me of a related question about the quality of urine specimens for human diagnostics. I'm wondering if you might have the possibility to improve the collection of the horse urine, so that you avoid the complexity the bacteria are causing for you. Remember that humans are often asked to provide 'midstream urine' samples, meaning that the patient is supposed to avoid collecting the first portion of the urine -- that first portion is often be contaminated with bacteria. I would imagine the same 'first urine contamination' problem exists in your equine samples. Is it possible to collect midstream urine only?
Related: human clinical urine samples often get antibacterial additives mixed into them immediately after collection, to retard bacterial growth. Is this a possibility for you?
Seems like it will be easier to reduce the bacterial presence in the first place, rather than coping with 'bad' samples later.
Do you have any clue about the size of your target DNA and the size range for contaminant DNA from bacterial cells? If you know the size, you can use gel extraction kit for purification of your target DNA.
Dear all,
I worked strongly in the field of forensic DNA, and it is the interesting field for me...
when you do in this field, the first thing to do is to make a DNA profile for you and fixing it on the notice table in the lab to compare any yielded profile with your one to dxclude cross contamination with exogenous DNA during any of mistaken error through performance daily work..
it is adivised to take precautions like, gloves, mask, lab coat snd etc...
on the other hand, amplificaion of human specific short tsndem repeats (STR) during real time PCR and apecific probe will help in differentiation of target from non target DNA priofile..,
best rdgard
Professor Dr.Mushtak T.S.Al-Ouqaili
Thanks to all! Actually I'm trying to amplify horse DNA to get a full genetic profile.
Specific amplicon (microsatellite) ranging from 90bp to 250bp and they're obtained using a specific kit (17-plex reaction, 34 primer all in one). The aspecific obtained are sadly part of the electropherogram so that is impossibile to discriminate what's the horse genetic profile as the aspecific fall exactly within the specific horse panel.
Craig, I don't know if it's possible to get midstream urine samples, but this could be useful indeed. Concerning the antibacterial additives I'm not sure it could be done as the sample is primarily collected for doping agents detection. Just in case, do you know where to get them and how to use them?
Thanks
The vendors (BD, Greiner, etc) of clinical diagnostics collection/transport tubes should have online about what they use in their products. These tubes have the chemicals already in them, so the urine is preserved as soon as it is collected.
For microbiology testing, there are well-defined clinical guidelines for urine collection and handling -- see, for example, http://www.specimencare.com/main.aspx?cat=711&id=6235 -- which has links to other resources also.
There is actually quite a bit in the literature about urine preservation chemistries. It is obviously a critical goal to have preservative chemistries that do not interfere with your target analytes (be they natural biochemicals, doping agents, metabolites of drug use, etc). You might find this document particularly useful - maybe some of your doping analytes are listed?: https://www.mayomedicallaboratories.com/it-mmfiles/Urine_Preservatives-Collection_and_Transportation_for_24-Hour_Urine_Specimens.pdf
You might focus on glucose as a surrogate for 'bacterial control', given that glucose would be food and thus depleted if bacterial growth was not stopped.
Thanks Craig though I think it's not a a matter of inhibition of the bacterial growth rather than of coextraction of bacterial DNA along with the equine one. The fact is that when you extract DNA you also extract bacterial DNA even if they're dead or their growth is blocked. I think the most valuable strategy could be the midstream urine samples as only in this case you can lower the bacteria levels unless you can use something able to digest bacterial DNA.
- Badges
- Science topic
- Similar topics
- Automotive Engineering
- Machines