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Thread: Questions about qPCR

  1. #1
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    Questions about qPCR

    Hello brewers and QC people,

    As we expand from 10k bbl/year last year, 25k this year, and 45k next year, we're going to need some better micro checks. I am pricing out the differences in cost between plating and qPCR. I have a very extensive background in qPCR, but was always using mammalian cell kits. If any of you currently use qPCR, what lysis kits do you use for yeast/hopefully not but possible bacteria, and how do you quantify it? Do you have a ratio from Ct to CFU?

    I think qPCR is the route to go, because it's a 40 min answer instead of 5 days. It's also a lot less labor. The capital cost is a lot though. I just need to find the right kits. I have primers for all the hop resistant strains already. halp!

    Cheers!
    Last edited by PCQC; 11-06-2015 at 08:11 AM.
    Peter Cronin
    Senior Quality Analyst
    AleSmith Brewing Company

  2. #2
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    Not a microbiologist myself, but fortunately I married someone much smarter and cooler than me. So I asked my PhD genomist wife about it. She pointed this out: http://invisiblesentinel.com/veriflo...lactobacillus/ Might be right up your alley.
    Russell Everett
    Co-Founder / Head Brewer
    Bainbridge Island Brewing
    Bainbridge Island, WA

  3. #3
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    Quote Originally Posted by Bainbridge View Post
    Not a microbiologist myself, but fortunately I married someone much smarter and cooler than me. So I asked my PhD genomist wife about it. She pointed this out: http://invisiblesentinel.com/veriflo...lactobacillus/ Might be right up your alley.
    Thanks! Yes I've heard of them and even had a demo done but I'm a firm believer in more open protocols. I can easily come up with my own primers and reagents for the qPCR whereas Invisible Sentinel is using proprietary reagents. Also, at $25 per reaction, I think it's way too overpriced. I worked in biotech and most companies lose money selling you the capital equipment, but make all their profit on kits for the equipment. I can't use a competitors kits with Invisible Sentinel. It's a good product if you don't care about cost and you want a quick answer.
    Peter Cronin
    Senior Quality Analyst
    AleSmith Brewing Company

  4. #4
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    Fair enough! I also looped in a friend who's a yeast genomics professor. FWIW both of them wondered why you'd bother with qPCR rather than just PCR. She said "Yeah, PCR beats plating. I agree qPCR seems like overkill unless you have bad/nonspecific primers that eventually amplify from anything. Gels are so easy to run that high schools routinely do them.
    Or maybe there's a CFU count that's tolerable and everything is always contaminated to some degree. That makes more sense."
    Russell Everett
    Co-Founder / Head Brewer
    Bainbridge Island Brewing
    Bainbridge Island, WA

  5. #5
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    PCR seems like an excellent way to get yourself really tied up in knots by false positives. I'm thinking about the recent hoopla about human DNA in hot-dogs--of course there's human DNA in hot-dogs--humans processed them (good luck finding a sample of anything on the surface of the Earth that does not have traces of human DNA)!

    qPCR might be a little less prone to false positives... or not. There's a lot of amplification in either, IIRC (not a geneticist, don't play one on TV, just try to keep up).

    Plating is pretty sweet for getting an idea of levels of infection--one organism, one colony.
    Timm Turrentine

    Brewerywright,
    Terminal Gravity Brewing,
    Enterprise. Oregon.

  6. #6
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    Quote Originally Posted by TGTimm View Post
    PCR seems like an excellent way to get yourself really tied up in knots by false positives. I'm thinking about the recent hoopla about human DNA in hot-dogs--of course there's human DNA in hot-dogs--humans processed them (good luck finding a sample of anything on the surface of the Earth that does not have traces of human DNA)!

    qPCR might be a little less prone to false positives... or not. There's a lot of amplification in either, IIRC (not a geneticist, don't play one on TV, just try to keep up).

    Plating is pretty sweet for getting an idea of levels of infection--one organism, one colony.
    Point well taken. DNA is the molecule of life for a reason - it's ridiculously stable. Haha. I'm hoping that with vigorous fermentation though, there's enough RNases and DNases floating around getting rid of those guys so that if DNA and RNA are present, then you really do have a problem because something is around that is still possibly active or alive or was recently.

    This is new territory and I'd like to at least try it out. I come from a biotech and academic background so I really want to bring in the latest equipment into the science of brewing.
    Peter Cronin
    Senior Quality Analyst
    AleSmith Brewing Company

  7. #7
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    Quote Originally Posted by Bainbridge View Post
    Fair enough! I also looped in a friend who's a yeast genomics professor. FWIW both of them wondered why you'd bother with qPCR rather than just PCR. She said "Yeah, PCR beats plating. I agree qPCR seems like overkill unless you have bad/nonspecific primers that eventually amplify from anything. Gels are so easy to run that high schools routinely do them.
    Or maybe there's a CFU count that's tolerable and everything is always contaminated to some degree. That makes more sense."
    True. Gels would be easier. I like the scalability of qPCR, there are a lot more things I can do. Also, I'd want to get the amplicons sequenced during primer testing if I was just doing PCR and then running a gel. That isn't so expensive nowadays at all, but that's how my old lab validated primer specificity. With qPCR I always looked at primer specificity using a melting step post-PCR and then I could see the melting temp of the amplicon. If it matched up with my calculated melt temp and only had one melt-temp peak, I know it is what I wanted without validating. I think with Open qPCR by Chai, I can get a machine for $3,000 and get a crap ton of good data. qPCR isn't that expensive anymore. Thanks for your reply!
    Peter Cronin
    Senior Quality Analyst
    AleSmith Brewing Company

  8. #8
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    Hi Peter,

    We've found BioTecon's foodproof StarPrep Four extraction kit to perform the best for extracting yeast DNA. For bacteria, we use the extraction kit that comes with the PIKA Weinhenstehpan LP PCR screening kit (simple detergent-based lysis + heat). We simply use this as a +/- test -- we haven't correlated CFU with Ct. You could, but do you care if there's 20 or 200 CFU? Either way you have a problem!

    I'm curious how you plan to design an assay with your primers / where you got your primer sequences from (if you're willing to share). The consumables are quite expensive...We got a good deal on a StepOne Plus from Life Technologies / Thermo Fisher. It was the most reasonably-priced open-source instrument. If you feign interest in their kits, maybe they'll cut you a deal as well! You could try to buy used, as well.

    Happy to chat more off-line,

    -Kendra

  9. #9
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    PCR Primers

    Sorry to revive a slightly older thread.... PCQC, where did you get/develop those primers? I'm looking to do some preliminary work with PCR myself, but haven't the skill to know how to develop the primers specific enough for the usual culprits. Any willingness to share would be greatly appreciated!

  10. #10
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    Quote Originally Posted by LAZPirate View Post
    Sorry to revive a slightly older thread.... PCQC, where did you get/develop those primers? I'm looking to do some preliminary work with PCR myself, but haven't the skill to know how to develop the primers specific enough for the usual culprits. Any willingness to share would be greatly appreciated!
    I will be starting a blog about getting brewing quality into the modern era of PCR and other new and newly cheap techniques. The primers I got from the following articles:

    "Isolation of a Hop-Sensitive Variant of Lactobacillus lindneri and Identification of Genetic Markers for Beer Spoilage Ability of Lactic Acid Bacteria"

    "Development of a PCR Method for Detection of Saccharomyces cerevisiae in Brewery Rinse Water"

    "Characterization of a Highly Hop-Resistant Lactobacillus brevis Strain Lacking Hop Transport"

    "A New and Rapid Method for Determination of Beer-Spoilage
    Ability of Lactobacilli


    Please excuse me for giving just titles and not authors - I just don't have time to do a full article reference format.

    I will eventually make my own primers, but right now I'm working with a company I used to work with back when I was in neuroscience. They are beer lovers and made a wild yeast and bacteria PCR. It's regualr PCR, not qPCR so you need to run a gel. Also it's go-no go. If you get a band in the gel, you have some wild yeast, but they put a bunch of primers in the reaction so you don't know what strain of yeast. The same goes for bacteria - they just put in a bunch of wort/beer spoiling bacteria-specific primers in one reaction. It's a good starting point though.
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    Peter Cronin
    Senior Quality Analyst
    AleSmith Brewing Company

  11. #11
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    Peter, I wanted to follow up on your efforts with PCR and how it has turned out. A go/no-go with a basic PCR may be enough for our current purposes.

    In addition, it sounds like a couple folks are using PCR already. I am curious to know how you are using it - specific checks on certain critical control points (e.g. propagation), checking all fermenters, packaging checks, etc. Are you using it to back up standard plating methods?

    Putting aside the capital expense, the price-per-check isn't cheap. I am curious to know how those who are using it are maximizing it.

    I would love to just swing into it with a One Step or Lightcycler and start using the non-proprietary kits, but I am working on making the costs work for the folks who cut the checks.

  12. #12
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    Lightbulb PCR primers

    Hello guys,

    My name is Marcio Buffolo and I'm Head Brewer/Scientist at Shades of Pale Brewing - UT.

    We are having some trouble with contamination due to Diastaticus. So, in order to avoid 5 days test, we are moving to PCR. I'm biologist and I have been working in molecular biology lab for more than 8 yeasrs. Our budget is short, so we have a Thermocycler and a Ethidium bromide system. We are going to regular PCR and gel. Do hyou guys have any good DNa extration protocol, Primer sequences and or full protocols for this type of tests?
    I'm doing a troubleshooting as well, so if I there is any thing that I can help, more than welcome to contact. buffolo.mATgmail.com

  13. #13
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    Hi,

    I was wondering what bacteria and wild yeast you guys are looking at? We're in the process of switching to qPCR as well.

    Thanks,

    Wendy

  14. #14
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    I'm trying to develop some primers to use in regular PCR (PCR/gel) to identify diastaticus, Brett and LAB's. The idea is developing primers with different sizes where i can include all in one reaction and and run a gel. I have more than 8 years of lab experience and 5 years of brewing (2 as head brewer). Medium plates are great, but takes more than 4 days to get good results. PCR take me 5 hours total.

    I will, for now, avoid qPCR. The DNA molecules are extreamally stable and there is a chance for false positive due the high detection levles. For now, PCR is our target.

  15. #15
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    Is anyone aware of a commercial multiplex real time PCR kit which has limits of detection comparable to GeneDisc or Invisible Sentinel? I've found a one-well test which detects lacto/pedio/megasphera/pectinatus for $15, but the detection limits aren't quite good enough to use without nutrient enrichement.

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