Using PCR to Identify Contamination in Beer – Part 3

Note to my readers: This technique does not work as well as hoped, for reasons discussed below. I am, however, completing this series as I get a lot of questions about it. While the method isn't perfect, it may be sufficient for some brewers. This method works by detecting diastaticus yeast via the presence of the Sta1 gene. As it turns out, simply detecting the presence of this gene is not enough to determine whether a strain is truly diastatic as there are many strains that have the Sta1 gene which are not diastatic - i.e. they don't super-attenuate the wort. This appears to be due to mutations in the promotor region (the part of the gene which turns the gene on and off) that prevents it from being using by the yeast. 
                                      -Bryan, Dec 4th, 2019

This is the third and final post in a my series on using PCR to detect contamination in beer. The impetus for this mini-series was a lawsuit launched against White Labs for contamination of commercial pitches with Saccharomyces cerevesia variant diastaticus, the most recent in a series of contamination issues at White labs. Note that this method is flawed, with these issues discussed in-depth at the end of this post.

In this post I will go over the method I have developed (based on methods published by others). The these are all based on PCR; a method of “copying” specific stretches of DNA isolated from fermenting beer, bottle dregs, a colony from a plate, or yeast slurry. The presence of the amplified DNA is then detected by gel electrophoresis. Three sub-tests are performed; the first is a positive control which will produce a band of 800-850 bp in size (for Saccharomyces), although other yeasts can give different sizes (350-900 bp). The diastaticus test, which detects yeast containing a gene that allows the yeast to super-attenuate beer (e.g. saison-style yeasts) that produces a band of 868 bp, and the bacterial test that produces a band of ~1500 bp, thus identifying the presence of any contaminating bacteria.

PCR for Detecting Contamination in Beer: 

• Part 1 – How It Works
• Part 2 – Equipment Required & Cost Breakdown
• Part 3 – The Method Itself and a Demonstration

DNA Isolation

The first step is to isolate the DNA from the potentially contaminated beer; this can be done using fermenting beer, purified colonies from a plate, bottle dregs, and from yeast slurry. The process is the same for all of these sources:

1. Collect an appropriate amount of yeast into a 1.5ml mirocentrifuge tube:
   • Fermenting beer: ~1 ml of beer.
   • Finished beer: up to 50 ml of beer (centrifuged multiple times to concentrate).
   • Slurry: ~50 ul of pure slurry (suspend in 1 ml of water).
   • Colony from plate: 1 medium-sized, or 2 small, colonies (suspend in 1 ml of water).
2. Pellet the yeast by centrifugation, using the maximum speed of your centrifuge until all cells are pelleted (generally 1 min):
   • You should end up with a yeast pellet the size of a BB (~5 mm, see above image).
   • If your pellet is larger than this, resuspend in water and remove a portion of the suspension to reduce the pellet size, then re-pellet.
3. Once you are certain the correct amount of yeast has been collected, remove all of the supernatant and resuspend in sterile distilled water. Then re-pellet the yeast as per step 2 and remove as much of the water as possible.
4. Using a 1 ml pipette, suspend the pellet in 200 ul of instagene matrix. Vortex for 15-30 seconds to completely break up the pellet.
5. Heat at 50C for 30 min.
6. Vortex for 1 min at maximum vortexer speed.
7. Heat at 100C (or as close to boiling as possible) for 5 min, then vortex at maximum speed for 1 min.
8. Centrifuge for 1 min at the maximum speed of your centrifuge to pellet the instagene matrix and cell debris; the supernatant contains your DNA.
9. The DNA can be used immediately, or the tube can be frozen in a deep freeze for several days prior to further use.

PCR Reaction

Prepare individual reaction tubes for each PCR test (positive control, diastaticus contamination test, and the bacteria contamination test):

For the positive control (detection of all yeasts):

Into a 200 ul PCR tube, add:

• 9.4 ul of sterile distilled water
• 2ul of 10X Tsg polymerase reaction buffer
• 0.2 ul of 10mM nucleotide solution
• 0.2 ul of Tsg polymerase
• 0.2 ul each of the ITS1 and ITS4 primers (pre-diluted to 100 uM)

For the diastaticus test:

• Same mixture as above, but replace the ITS1/4 primers with the SD-5A/SD-6B primers.

For the bacteria test (if included):

• Same mixture as above, but replace the ITS1/4 primers with the pA/pH primers.

Once reaction tubes are prepared:

1. To each reaction add 8 ul of the DNA isolated above (supernatant from the instagene process) and pipette up/down repeatedly to mix.
2. Seal the PCR tubes and place into your thermocycler.
3. Run the following PCR program:
   1. 2 min at 95C
   2. 30 sec at 95C (denature)
   3. 30 sec at 50C (anneal)
   4. 90 sec at 72C (elongate)
   5. Repeat steps #2 to #4 (denature→anneal→elongate) 35 times*

* Note: The number of cycles (step 5) can be increased to 40 or even 45 cycles to increase sensitivity, but you are also more likely to generate a false-positive due to trace amounts of contaminating DNA from the environment.

DNA Gel Electrophoresis

Prepare a 1.5% agarose gel; volumes may need to be adjusted depending on the size of your gel. This assumes a standard (30 ml) mini-gel:

1. In a small (250 ml) flask, measure out 30 ml of 1X concentration TAE buffer, and then add 0.45 g of agarose.
2. Microwave until it boils; immediately remove from microwave and swirl to mix. Repeat 3-4 times, until agarose is completely dissolved.
3. Add 3 ul of a 10 mg/ml solution of ethidium bromide, and swirl gently to mix.
4. Pour the gel into the gel tray (follow your manufacturers instructions) and insert comb; let solidify (~20 min).
5. Place gel into your electrophoresis apparatus and fill with 0.5X TAE buffer, as per manufacturers instructions.

Prepare & load your DNA samples:

1. To each 20 ul PCR reaction add 3 ul of 6X DNA loading dye.
2. Remove the comb from the gel.
3. Place 20 ul of DNA/loading dye mixture into each well, using 1 well per sample.
4. Load 3-6 ul of DNA ladder into an empty well on one side of the sample.
5. Run the DNA gel at 100V for 40-80 min; be careful not to run your DNA sample off of the end of the gel.

Imaging the Gel

1. Put on UV-protective goggles or a UV-protective face-mask.
2. Place the DNA gel onto the transilluminator and turn on the transilluminator.
3. Take a picture of the gel.

Interpreting the Test

Interpretation of the test is straight forward; the PCR reaction should produce DNA bands of a known size if the tested organism is present, and the band will be absent if the organism is not present. Contamination is indicated by the presence of a diastatic or bacteria DNA band in beers where these organisms are not expected.

The positive control  tests for the presence of any eukaryotic DNA (e.g. yeast/fungal DNA, or any contaminating human, fly, plant, etc, DNA). The size of this band can vary, but should be 800-850 bp for Saccharomyces. Other species will give bands of differing sizes (350-900 bp), so you may see a differing size band if fermenting with something other than conventional brewers yeast. Multiple bands may be present if multiple yeasts are used. As designed, you shouldn’t get reactions from plant DNA (e.g. malt or hops), although you may see amplification of this type of DNA if you use extra PCR cycles to increase sensitivity.

Interpreting the Positive Control:

• 1 or more bands present – your DNA isolation and PCR reaction worked.
• No bands – test failed, or only bacteria are present. If yeast was expected, discard all samples and repeat the test from the beginning.

The diastaticus test tests for the presence of contamination with diastatic Saccharomyces. This test specifically looks for the presence of the STA1 gene, which is a secreted diastase enzyme that breaks down starches and dextrans into glucose that the yeast then ferments. Yeast with this gene have the potential to be diastatic, although work by Kristoffer Krogerus (link only available to milk-the-funk members) indicates that some strains may have a STA1 gene that is otherwise non-functional. As such, a positive test should be interpreted as the beer most likely contains a diastatic strain. The resulting DNA band should be ~860 bp.

Interpreting the diastaticus test:

• Band ~860 bp in size: Beer most likely contains a diastatic strain.
• No band: Beer is free of diastatic yeast/diastaticus is below detection.

The bacteria test tests for the presence of bacterial contamination by looking for genes encoding the bacterial ribosome; a universal and minimally variable cellular structure found in all life forms. The specific primers use will amplify a region of the 16S ribosome found in all bacteria, but which is absent in Eukaryota (yeast, plants, humans) or archeans (bacteria-like organisms that grow in places like hot springs). It should be noted that this test does not tell you what bacteria are present; it only indicates the presence of bacteria.

Interpreting the bacterial test:

• Band ~1500 bp in size: bacterial are present.
• No band: no bacteria are present.

Sample (Contamination) Test

Below is a DNA gel mimicking the results of a test run of a mixed fermentation containing yeast and bacteria; on the left a mixed-ferment containing a non-diastatic yeast is shown, on the right a mixed-ferment containing a diastatic yeast is shown. For simplicity I have not included the control reactions.

I have also multiplexed one of the PCRs by adding both the bacteria-detecting and diastaticus-detecting primers to the same PCR reaction. This is a method which can reduce your costs of performing this test, but it is critical you only do this using primers that produce bands of very different size, and with primers that don’t cross-react with each other. For example, you would not want to use the yeast and diastaticus primers together, as they both produce PCR products around the same size (850 bp).

The Issues

On the surface this method appears to work, so why have I been telling you that it failed. The problem with this approach was discovered by Escarpment Yeast Lab, who found that a positive result on this PCR test (e.g. identifying the Sta1 gene in a strain of yeast) did not accurately predict the diastatic capability of the yeast. Strains which lacked Sta1 were never diastatic, but there were strains with Sta1 that also were also not diastatic.

Escarpments experiments in this area can be found in the following three posts: Post 1, Post 2, Post 3.

In other words, a yeast has to have the Sta1 gene to be diastatic, but the presence of the Sta1 gene is not enough to confer diastatic capabilities on a yeast strain.

The reason behind this isn’t entirely clear, but for a portion of the strains with the Sta1 gene, but without diastatic activity, the reason appears to be a deletion in the promoter region of the Sta1 gene. In other words, these strains are missing the part of the Sta1 gene that is used to turn the gene on and off. So while the yeast have the genetic information to make the Sta1 gene, they lack the ability to “access” that genetic information and therefore are not diastatic.

The sensitivity of this method may also not be sufficient to identify the kind of low-level contamination that could lead to bottle over-carbonation.

What this ultimately means is that you could use the approach I show above to test for the presence of diastaticus yeast in your brewery. However, the results must be taken with a big grain of salt, as low-level contamination could be easily missed, and a positive result may overstate  the risk of diastatic activity in your brewery.