I'd recommend doing an additional filter step with .5um filter tubes after the lysis but before the thermal cycle. This should help with getting rid of any remaining debris. Do you add a chelator to the sample, like EDTA, to assist with non-specific binding? If you have excessive PCR cycles this can also amplify any non-specific binding that is occurring. For recovery of the lacto/pedio NBB and MRS are both great choices. I'd also recommend plating the sample on LMDA both aerobically and anaerobically.
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Kory Davis
QC and Propagation Specialist
The Brewing Science Institute
Woodland Park, Co
719-482-4895 ext. 3
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Original Message:
Sent: 04-12-2023 14:01
From: Anonymous Member
Subject: PCR issue with Hazy beers.
This message was posted by a user wishing to remain anonymous
We are currently enriching in MRS with roughly 48 hours of enrichment time. We do add less beer to the MRS than we do with our non-hazy beers. Before we do the lysis step, we clean-up extracellular debris with a low RPM spin down. We are currently using Bitoecon kits with a Roche lightcycler 480. We have confirmed with Biotecon that these are positive hits for lacto./pedio. We have not had any luck with culturing cells using traditional plating methods, hence the posting.
Original Message:
Sent: 04-12-2023 12:24
From: Kory Davis
Subject: PCR issue with Hazy beers.
Hello,
False positives are known to occur when there is excess material in a sample. Hazy beers, being what they are, are heavily loaded with lots of extra material. This could be throwing off your results. If you are not already, I would suggest doing an additional filter step to help reduce the quantity of material in your sample. .5um Eppendorf filter tubes work great. If I may ask, what platform are you using and are you performing a lysis step?
As far as enriching your sample to increase your detection threshold, MRS should get the job done.
Kory
------------------------------
Kory Davis
QC and Propagation Specialist
The Brewing Science Institute
Woodland Park, Co
719-482-4895 ext. 3
Original Message:
Sent: 04-11-2023 11:45
From: Anonymous Member
Subject: PCR issue with Hazy beers.
This message was posted by a user wishing to remain anonymous
My brewery is having a new PCR issue with false positives for Lactobacillus in Hazy beers. I sent beer to an outside lab and they did not detect any beer spoilers?
Anyone else experiencing this issue?
Also, what are people using for enrichment media? I have heard NBB, MRS and Fast Orange. Any suggestions from veteran micro people?