Bio-Rad VINEO™ Brettanomytest PCR Kit - Manual (Page 4)

• Negative
• Low population, controlled risk
• Critical population to be monitored
• Very high population, risk of production of volatile Phenols
The VINEO™ Brettanomytest PCR Kit can also be used in a qualitative
mode to confirm isolated colonies from a culture medium.
2 - PRINCIPLE BEHIND THE VINEO™ Brettanomytest PCR Kit
This test is based on amplification, detection and quantification of DNA
sequences using the real-time PCR technique. It uses primers and a DNA
probe that are specific to Brettanomyces bruxellensis. Detection and
analysis of the results are optimised for use with a thermocycler for
Bio-Rad real-time PCR such as MiniOpticon™, Chromo4™ or CFX96™.
During the PCR reaction, the primers will be hybridised in the target region
and will then - catalysed by the polymerase - lengthen in the 5'-3'
direction using the desoxynucleotide triphosphate (dNTPs) present in the
reagent mixture, thereby creating a complementary DNA sequence, called
an amplicon.
During PCR, oligonucleotidic probes that are specific to the target
sequence will be hybridised with the amplicons. These probes, which are
marked by fluorophores, only emit fluorescence when hybridisation takes
place. The probe that is linked to the target sequence of Brettanomyces
bruxellensis is marked by a specific fluorophore. Intensity of fluorescence
increases proportionally to the increase in quantity of amplification
products in the PCR tube. The fluorescence that is generated in this way
is directly measured by the optical module of the thermocycler.
A synthetic DNA called the "Internal control" is added to each reaction. It
is amplified at the same time as the target sequences of Brettanomyces
bruxellensis but detected by a probe marked with a second fluorophore. It
is used to bring out any reaction inhibiting phenomenon.
The software associated with the device calculates the relation between
the intensity of the fluorescence and the amplification cycle automatically.
This relation shows the presence or absence of Brettanomyces
bruxellensis in the sample.
The results obtained by amplification of this DNA are analysed by the
Opticon Monitor™ or CFX Manager™ IDE software that have been
programmed to carry out an automatic and quantitative analysis. An
interpretation of the risk linked to the presence of Brettanomyces
bruxellensis is then proposed to the user.
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