We need to extract pathogen DNA from faeces. However, the amount of the pathogen's DNA (a parasite) is likely to be very small.
So, in order to avoid false negative results, we should consider a relatively big amount of faeces for the extraction… with all the drawbacks that such an approach entails.
We already tried QiAmp Stool Mini Kit (Qiagen) on 220 mg of feces … but without success (probably due to the small amount of sample utilized).
Now we intend to utilize a bigger amount of fecal sample (5-20 gr).
In this view, we are looking for a method that allows to concentrate (in some way) the original sample.
With anticipated thanks to everyone will be able to help me and my team
I am a bit confused, pathogen as in a microbe or parasite like a worm.
In case of bug, standard stool dna extraction kits followed by deep sequencing on a next generation platform should be able to capture the pathogen of choice.
In case of a parasite like a worm, the easiest is to confirm the presence of its eggs in the stool vide microscopy.
Just curious what method are you using to detect presence/absence..is it QPCr or sequencing and what is your positive control for the technique.
We are studying a parasite, precisely Enterobius vermicularis (pinworm).
We tried to recover the eggs following different protocols (first of all Wisconsin flotation), but we have never observed them in microscopy.
However, some samples not showing eggs, were positive by PCR (a "double" PCR, using the same pair of primers for two reactions).
According to these results, we think that the DNA amplified is likely to be nucleic acid from exfoliated cells of the adult worm.
Our positive control is DNA extracted from positive scotch tests.
Looks like you are doing everything right so the only thing maybe is the PCR primers, does the ones from this paper help
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3122864/
Getting into the details, the female however is known to locate to across the intestine depending on the stage of matuarity so the sample collection would play a role is capturing a higher amount of the worm.
The easiest would be to just use the scotch test DNA itself, if your clinical protocol allows.
Interesting question indeed.
Allow me to clarify a few issues that I should point out first:
1) We designed new primers on our own and they showed to be specific only for E. vermicularis in pre-clinical studies (conducted using DNA recovered from positive scotch tests). So, we think they are not the impasse of the study.
2) Regarding the sample collection... This is the main issue. We need to detect E. vermicularis even if its DNA presence in the bowel is exigue.To do this, we need to "concentrate" (or something similar) all the nucleic acid from a large stool sample BEFORE the extraction.
3) We cannot use scotch test as diagnostic tool because of its low sensivity (about 20%). It's our gold-standard (together with the evidence of worms in the faeces).
Thank you for the interest and the help.
The only thing in that case is to make bead or column that will capture your DNA sequence of interest and use that for PCR amplification.
Thanks for the details.
Enjoyed the challenging question.
Can you suggest us something more specific? For example a kit..
Thank you for the interest and the help.
http://www.ncbi.nlm.nih.gov/pubmed/22237535
The above is the science behind the idea.
Here is a link on a kit but it works for microbes only
https://www.neb.com/products/e2612-nebnext-microbiome-dna-enrichment-kit
and another example using a different strategy.
http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0014004
Most welcome..do keep me posted on how things progress, I am intrigued by this scientific endeavour..
Good luck.
Not yet. We have just decided to process other samples using another kit, Power Soil DNA Isolation Kit (Mo Bio). Even if it is usually used for soil samples, it works properly also on faeces. Its protocol is very similar to Qiagen's one, but with the introduction of beads to fully distrupt cell membranes. These beads seem to be efficient also for parasite's eggs. So, using the faeces directly (and not the supernatant as we did) and processing them with this kit could be the best strategy to obtain as much DNA as possible. We have also found some papers to support this hypotesis. Hopefully, this will be the right time. I'll keep you updated.
It is a good kit, but has low yields compared to other kits but the quality of DNA is better and the steps are easier.
Good luck, look forward to hearing exciting results from you.
CHeers
Power Soil didn't work as it was expected to. We think that the problem is the disruption of faeces through beads. Now we are looking for a good machine to do this step properly.
I will update you as soon as we will have the first results.
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