Bio-Rad VINEO™ Brettanomytest PCR Kit - Manual (Page 10)

Interpretation of results obtained for the samples:
**: In the event of a null value for a sample and its internal control (Ct = N/A), the analysis must be done
again with the diluted DNA sample (diluted to 1/5
th or to 1/10th for example).
***: A value less than 10 may be obtained; check whether the curve as raw data shows an aspect that
is characteristic of exponential amplification (flat base line and regular increase in fluorescence followed
by a plateau). If the observed curve is correct, the sample for the presence of Brettanomyces
bruxellensis may be considered to be positive. Otherwise, the interpretation of the result depends on
the value of the internal control, as is explained in the previous paragraphs.
12 - CONFIRMATION PROTOCOL FROM ISOLATED COLONIES
You can use the VINEO™ Brettanomytest PCR Kit to confirm isolated
colonies on agarose gel.
1. Puncture an isolated colony, whether it comes from a selective medium or
not, using a toothpick or a 1 μl piercing instrument, or any other
consumable adapted to this purpose (a pipette cone for example).
2. Resuspend the colony in 100 μl of R2 in an Eppendorf tube (it is possible
to resuspend the colony in steril physiological water, PCR efficiency can
then be affected). Homogenise the suspension by means of a vortex.
3. Put 5 μl of suspension in 45 μl of PCR reagent mixture (cf. part 9.2
Real-time PCR) and follow the rest of the VINEO™ Brettanomytest PCR
Kit method to obtain and interpret results. Note: only a qualitative
analysis is made from the PCR carried out on this suspension. The
signal obtained by PCR is often quite high because the quantity of cells
sampled and analysed by PCR using this method is quite significant.
13 - TEST PERFORMANCE AND VALIDATIONS
The VINEO™ Brettanomytest PCR Kit is specific for detecting
Brettanomyces bruxellensis. You can detect the yeast from:
• 500 CFU/mL of wine using the DNA extraction protocol described in
the VINEO™ Extract DNA Kit: for 1.8 mL for fermenting grape must or
a heavy wine during the wine-making or maturing process and
• 10 CFU/mL of wine according to the protocol starting from 45 mL for
slightly heavy wine, close to bottling or already bottled, the analysis of
which requires a high level of analytic sensitivity.
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Detection of Brettanomyces
bruxellensis (FAM)
Detection of the internal
control
Interpretation
Ct ≥ 10*** Not significant Positive
Ct = N/A Ct
HEX
≥ 28 Negative
Ct = N/A Ct = N/A Inhibition**