Qiagen stool DNA kit will be work well for extraction of metagenome (Combined genomes of all microbes) but in your case you are looking for specific DNA, you do either separate the desire organism(if possible) and then extract the DNA or from whole metagenomic DNA by organism specific PCR. You also mentioned that you got only 3% positive result  for your desire organism, but the proportion of any microbes in gut is depended on the other major occupant in the gut, may be fiber, polysaccharide degrading microbes constitute much more in the cattle gut than your desired organisms. 

Chances are the F hepatica eggs are not being broken down in your extraction method (and you may also be suffering from PCR inhibitors from the faeces). What kit/ method are you using currently?

Its been a couple of years since Ive done parasite DNA extractions (now working on bacteria in sputum! but used to extract DNA from parasite larvae from faeces) but some kind of mechanical (eg bead beating) disruption plus chemical/ enzyme (proteinase K?) should work. MoBio has kits called PowerSoil and PowerFeacal which may work well- old colleagues of mine at Moredun Research Institute may have more up-to-date information for you! Have you looked at the yield/ quality of DNA you currently recover from faeces?

Hi Alison,

I used the PowerFecal kit from MoBio actually, according to their protocol, but seems like it doesn't work that well for me. I tried various methods, including mixing the faecal samples with water 1:1 (according to some literature this works) and extracting it with the MoBio kit again and also with a tissue DNA extraction kit (Qiagen). I tried to do the beating for longer than suggested, still didn't work. Added an additional 95°C heating, still didn't work either. By the way, I haven't looked at the quality of the DNA, but directly ran PCR and very few samples were positive, so I assume that there was no target DNA in the extractions. I didn't want to use a DNA concentration measurement, since plant DNA and other DNAs are there too, but maybe I should check it anyway. Thanks for your recommendations!

Hi Abhijit,

The idea is to extract DNA without having to look for the parasites, and yes there is a lot of plant material, and I also think this is a problem with livestock faeces.

Maria,

Always frustrating when the kit you think should work (and was designed to work with your samples) best doesnt! 

Perhaps the DNA has co-extracted with faecal contaminants that inhibit the PCR reaction? Nanodrop profiles may help. Good luck.

Alison

Just one thought regarding the MoBio kit (and feel free to ignore if Im stating the obvious, I just know how frustrating it is to not get the DNA you expect from samples!)- what vortex adapter did you use? Did you keep the tubes horizontal whilst vortexing?

A student in our lab worked on molecular diagnostics of Fasciola hepatica and Fascioloides magna (a relative of F. hepatica) in stool samples. She tried several protocols and the best one was based on modified protocol of the QIAmp DNA Stool mini kit (Qiagen) - combined with homogenization using a bead beater - Mini-Beadbeater-1 (BioSpec) (1 min, 5800 g). Homogenization of the faecal sample containing eggs refined by filtration through a mesh was performed in ASL buffer contained in the kit. Incubation with proteinase K was performed for 24 hours. Depending on primers used, the sensitivity of PCR was as high as 1 egg per 1 ml of refined (filtered) sample. Hope this will help you. Good luck! Libor Mikeš

Hello Libor Mikeš,

Thank you for your suggestion, it sounds promising. Just a question, the faecal sample was refined by filtration through a mesh. Was the faecal sample homogenized in ASL buffer and then filtered through the mesh or was the faecal sample already filtered (by mixing with water/other liquid and passed through a mesh) and then the filtrate mixed with buffer ASL? It would be really helpful to know the ratios of faeces : ASL or water used.

Maria

Hi Allison,

Yes, I was thinking that PCR inhibitors got extracted too, so I will check the quality as you have suggested :)

As for the tubes, I vortexed them horizontally on a plate shaker or a vortex bed (I taped them securely) for like 15 minutes on maximum speed.

Hi Maria

Hello Maria, here is the whole well-working protocol (at least in our lab :-)

The stool sample was resuspended in water and filtered and washed through a mesh (ca. 1 mm) to remove an excess of ballast material (cellulose etc). After decantation, the supernatant was replaced by fresh water (performed three times). This will help you to get rid also of a large proportion of microorganisms. The final sediment (1 ml) was transfered into a 2 ml polypropylene tube (BioSpec) and 0.5 ml of glass beads (1 mm diameter) was added (BioSpec). Suspension was centrifuged (1 min 16000 x g), supernatant was removed and 1 ml of ASL was added to the sediment. Homogenization was performed on Mini-Beadbeater-1 (BioSpec) (1 min, 5800 g). Incubation followed for 10 min at 70 °C. The sample was centrifuged (1 min, 16000 g), supernatant transfered into a clean tube and mixed with ½ of InhibitEX® tablet (part of the kit). After centrifugation (1 min, 16000 g), the whole amount of supernatant was incubated at 70 °C with AL buffer and proteinase K (ratio 200:200:15) for 24 h. Finally, 96% ethanol was added (the same amount as of AL buffer) and transfered onto column... (see kit manual).

Libor Mikeš

Thank you for your detailed description Libor Mikeš! I really appreciate it.

Maria

Hi Maria!

I work with Angiostrongylus spp. In order to identify the species, I have to perform PCR reactions. The larvae are so difficult to break so, as other colleagues have already told you, you might need mechanical processes before or together with your extraction method. I use QIAamp Tissue and Blood DNA extraction for my purpose. Maybe you will need another kit which has a better purifying process, because of inhibitors present in faeces.

Here´s what I do: I put glasses beads in the samples together with the Lysis buffer and proteinase K (quantities recommended by the manufacturer) and vortex horizontally at maximum speed for, at least, 10 minutes. After that, I follow the extration steps.

It works really good for me. Other option in aquiring a lysis system, like Tissue Lyser, from Qiagen. They are great!

Hope to help!

Lilian

Not sure if you found the answer you are looking for, but the company I work offers a gastrointestinal pathogen panel, which includes a pre-treatment workflow prior to extraction that that is for DNA/RNA isolation from viruses, parasites, and bacteria from the same sample. Feel free to contact me for more information on the pre-treatment step.