How do you check for bacterial DNA contamination?

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mlolso3
mlolso3's picture
How do you check for bacterial DNA contamination?

I have been isolating DNA from buccal brushes for quite some time and I have just recently started isolating from brushes that have been sent to us from patients through the mail. These new samples have a much higher DNA content then the fresh samples that we take and freeze within an hour. I am worried that the excess DNA is from bacteria that grew during shipment of the brushes through the mail. These brushes were not dried before mailing so the combination of moisture and cells taken from the mouth left at R/T for days leads me to believe there might have been some bacterial growth. Is there a way that I could check for this? The only thing I can think of is to run a gel and look for DNA degradation which might indicate bacterial growth. I looked at a couple samples under the microscope before lysing the cells and all I saw were epithelial cells.

Omai
Omai's picture
Are the buccal brushes are

Are the buccal brushes are supposed to be 100% bacteria free when they arrive to you? If so, you could do PCR on the sample using primers specific to gene encoding 16s rRNA. This is bacterial specific ribosomal RNA. The sample should be negative if there is no bacteria present. This will only work if your noncontaminated sample is 100% bacteria free. Its PCR, so it will amplify very small amounts of DNA.

Omai

Here are some primer sequences and a protocol.
Hope this helps.

http://www.protocol-online.org/biology-forums/posts/13485.html

mlolso3
mlolso3's picture
That is a good idea,

That is a good idea, unfortunately even the fresh samples should have a little bacteria present since they have come from the mouth.

Omai
Omai's picture
http://www.ncbi.nlm.nih.gov

http://www.ncbi.nlm.nih.gov/pmc/articles/PMC321263/

In this paper they use qPCR and the 16s rRNA to quantify the amount of bacteria found in a fecal sample. THis technique should work on your buccal brush sample. Take a control swab from your own mouth to quantify the number of bacteria, and then compare it to you samples sent through the mail. You should be able to determine whether there has been bacterial growth on your sample.

Omai