Detection of Brettanomyces in Wine by SYBR Green PCR
This protocol has been developed from the paper by Phister and Mills (2003), using
Dekkera bruxellensis (ATCC 34448; syn. Brettanomyces intermedius) as positive control.
The minimum reliably detectable number of cells per 50 mL of wine was found to be
5000 cells (=100 cells/ml), although it will inconsistently give positive reactions with cell
densities as low as 10 cells/ml of wine (Amsden and Vincelli, unpubl.). These primers
did not amplify the wine fermenting yeasts Saccharomyces cerevisiae cerevisiae (Phister
and Mills (2003) nor Saccharomyces bayanus (Amsden and Vincelli, unpubl.).
Sample preparation: 50 ml of wine, filtered through sterile Mira Cloth (0.22 µm). Wash
retentate into a sterile 15 ml Corning tube using 1 mL sterile Nanopure water. Transfer
this suspension to a 1.5 mL microcentrifuge tube and use immediately in PCR reaction or
store at -20⁰C. Be certain to gently mix suspension IMMEDIATELY prior to adding
sample aliquot into the PCR reaction tube.
PCR Master Mix (per 25 µl reaction):
BSA @1mg/ml*
4.5 µl
^
Fast SYBR Green Master Mix
12.5 µl
Primer DBRUXF, 5 µM
4.5 µl
Primer DBRUXR, 5 µM
1.5 µl
23 µl aliquots/rxn
Add 2.0 µl filtered cells or controls as noted below.
*Sigma Cat. No. A-7030, filter-sterilized.
^
Fast SYBR Green Master Mix, Applied BioSystems
Primers
Prepare to a concentration of 5 µM (5 pmol/µl) in TNE buffer:
DBRUXF 5’-GGATGGGTGCACCTGGTTTACAC-3’
DBRUXR 5‘-GAAGGGCCACATTCACGAACCCCG-3’
Controls:
Negative control #1: 2 µl Nanopure water
Positive control #1: 2 µl of Dekkera bruxellensis (ATCC 34448) DNA extract, 1.0
ng/µL.
Positive control #2: 2 µl of Dekkera bruxellensis (ATCC 34448) plus 2 µl of unknown
sample. If results show evidence of PCR inhibition, retest
unknown sample at 10-fold dilution and 100-fold dilution.
Cycling Protocol run on Cepheid SmartCycler (default ramp rate)
Name of Protocol: Fast Bretts
1. 95ºC for 300 sec
2. 40 cycles of: 95ºC for 3 sec off default ramp rate
69ºC for 30 sec on default ramp rate
3. Melt Curve
60⁰C to 95⁰C at 0.2 deg. / sec.
Duration of protocol: 45 min
Expected Results
Amplicons are 77 bp with an expected Tm of 79.4 - 81.5.
In the event that confirmation of PCR product is desired, an easy-to-use dot blot
assay was developed. The use of a digoxigenin-labeled probe specific to D.
bruxellensis and internal to the DBRUXF and DBRUXR primer pair enables
confirmation of the amplicon product, whereas direct sequencing of the amplicon is
difficult due to its extremely small size.
Digoxigenin Dot Blot for Dekkera bruxellensis using Brett Probe 1
Brett Probe 1: 5’-GCCAGCATCGGTTCTGGGAGCCATATAC/3DigoxN/ -3
Dilute in TE 5 ng/µl.
Hybridization Solution: 0.125 M NaHPO4, pH 6.2, 7% SDS, and 1 mM EDTA
Dot Blot Procedure:
Membrane size - Allow a 1 cm2 area per PCR reaction on the membrane.
1. Spot 1 µl of each PCR amplicon onto positively-charged nitrocellulose
membrane. Be certain to include positive and negative control reactions.
2. Denature the DNA by washing the membrane in 0.5 N NaOH at room
temperature for 10 minutes.
3. Do a quick rinse of the membrane in 2x SSC at room temperature, followed by a
10 minute incubation in fresh 2x SSC.
4. Place membrane on a paper towel and “fix” the DNA to the still damp membrane
using a UV crosslinker at 120 kjoules/cm2; allow to air dry.
a. Note: if a Hold step is necessary, place membrane between paper towels
and place in a 55⁰C oven until ready to continue.
5. Pre-hybridization: Pre-warm 10 ml of hybridization solution without Brett
Probe 1 in a 65⁰C hybridization oven. Add membrane and incubate for 15
minutes at 65 ⁰C.
6. Hybridization steps:
a. Pre-heat the tube containing the Brett Probe 1 to 65⁰C for 5 minutes to
eliminate secondary structure. Decant the pre-hybridization solution and
add 5 ml of fresh, pre-warmed hybridization solution. Add 10 µl of Brett
Probe 1 directly to the hybridization vessel, place back into the oven, and
hybridize overnight at 65⁰C.
b. Next morning, rinse the excess probe from the vessel by the addition of
60 ml of room temperature 2xSSC containing 0.1% SDS and incubating
in the oven 10 minutes at 65⁰C.
c. Wash at low stringency 2 times with 60 ml of 2x SSC + 0.1 % ,
incubating at 65⁰C each time for 10 minutes each wash.
d. Blot membrane with a kimwipe and wrap the membrane in plastic wrap.
e. At this point the membrane may be used immediately for detection using
a Digoxigenin Dot Blot kit (Roche DIG Nucleic Acid Detection Kit, #11
175 041 910) OR stored at 4⁰C until ready to do the detection steps.
Follow kit directions.
7. Detection steps
a. Place hybridized membrane into a sterile tube (Corning 50 ml) for antiDIG binding.
b. Rinse briefly with Washing buffer (see kit directions), followed
immediately with a 30 minute incubation in 20 ml of 1x Blocking
Solution (see kit) (2 ml of Blocking Reagent added to 18 ml of Maleic
Acid buffer) Place tube on a rocking platform.
c. Decant Blocking solution and add 20 ml of Antibody Solution (anti-DIG
conjugate @ 1/5000 in 1x Blocking Solution. Incubate for 30 minutes,
room temperature, rocking:
d. Wash twice in 40 ml of Washing Buffer, rocking 15 minutes each wash.
e. Equilibrate pH with a 5 minute incubation in 20 ml of Detection Buffer
(see kit).
8. Color reaction steps:
a. Use a cleaned, dedicated plastic box, darkened with foil to protect from
light.
b. If NBT/BCIP has precipitate in tube, warn to 37⁰C briefly and vortex to
mix. NBT/BCIP may subsequently be stored in darkened tube at 4⁰C.
c. Add 200 µl of NBT/BCIP to 10 ml of Detection Buffer poured into the
detection box to which the membrane has been placed.
d. Leave the box undisturbed (NO ROCKING!) throughout color
development.
e. Two to 2 ½ hours have been found sufficient for detection of as little as 5
ng of Dekkera bruxellensis amplicon.
f. Stop the color reaction with the addition of 50 ml TE, pH 8.
g. The blot may be photographed under white light.
.
References:
Stender, Henrik et al. Identification of Dekkera bruxellensis (Brettanomyces) from wine
by Fluorescence In Situ Hybridation Using Peptide Nucleic Acid Probes. Appl. Environ.
Microbiol. 2001 February; 67(2): 938-941.
Phister, Trevor G. and David A. Mills. Real-time PCR Assay for Detection and
Enumeration of Dekkera bruxellensis in Wines. Appl. and Environ. Microbiol. Dec.
2003, p. 7430-7434.