I am extracting bacterial DNA from food samples for 16S sequencing; processed foods, ready to eat products, meat etc. We have a problem with high levels of host DNA, especially soy DNA, which often interferes with the sequencing and assigning taxonomy.
Does anyone have a optimal protocol or kit recommendation for reducing this problem?
These two publications can be useful for you since they describe protocols for reducing, for example, Sus scrofa DNA in the samples.
Boessenkool S., Epp L.S., Haile J., Bellemain E., Edwards M., Coissac E., Brochmann C. 2012. Blocking human contaminant DNA during PCR allows amplification of rare mammal species from sedimentary ancient DNA. ‒ Molecular Ecology 21, No 8: 1806-1815. doi: 10.1111/j.1365-294X.2011.05306.x.
Calvignac-Spencer S., Merkel K., Kutzner N., Kuhl H., Boesch C., Kappeler P.M., Leendertz F.H. 2013. Carrion fly-derived DNA as a tool for comprehensive and cost-effective assessment of mammalian biodiversity. – Molecular Ecology 22, No 4: 915–924. doi: 10.1111/mec.12183.
Hi, Through the experiences of the appointment, The 16 S rRNA or16 S rDNA not have many sediment. I thought , You can use specialized primers of each any bacteria e.g.
- Detection of Listeria monocytogenes bacteria in four types of milk using PCR or
- detection of listeria monocytogenes HlyA gene in meat samples by Real-Time PCR in Erbil city
And use promege kit is very best for DNA bacteria isolated
Hi,
I recommend using Dneasy power Food kit for extracting the total genomic DNA, Its very recommended for processed food and has the ability to reduce any PCR inhibitors found in your samples. I found it very useful with Milk samples and the obtained DNA was enough and clean for the downstream sequencing
You can contact the manufacturer for kit sample to try.
another trick is try to minimize your sample volume.
Good luck
Hi,
one or combination of the below mentioned steps may help you.
1) As suggested in the earlier replies you can first try out various kits for DNA isolation.
2) Increase the stringency of PCR reaction i.e, try different concentrations of Mgcl2, primers and also try playing around with DNA concentrations also.
3) You can add some additives to the PCR reactions or tags to your PCR primers which may also increase the specificity of PCR amplification.
Hope this helps,
All the Best..
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