Bio-Rad VINEO Brettanomytest PCR Kit User Manual [en, es, fr, it]

VINEO™Brettanomytest PCR Kit

Ref.: 354-8101

Instructions

Test to detect and quantify Brettanomyces bruxellensis using real-time PCR

TABLE OF CONTENTS

1 - INTRODUCTION

2 - PRINCIPLE BEHIND THE VINEO™ Brettanomytest PCR Kit

3 - KIT COMPOSITION

4 - VALIDITY AND PRESERVATION

5 - REQUIRED EQUIPMENT THAT IS NOT PROVIDED

6 - PRECAUTIONS AND RECOMMENDATIONS

7 - SAMPLING AND TRANSPORT OF SAMPLES

8 - DNA EXTRACTION

9 - REAL-TIME PCR

10 - ANALYSIS OF THE DATA

11 - INTERPRETATION OF THE RESULTS

12 - CONFIRMATION PROTOCOL FROM ISOLATED COLONIES

13 - TEST PERFORMANCE AND VALIDATIONS

APPENDIX A - PCR AMPLIFICATION REAGENT MIXTURE

PREPARATION TABLE

APPENDIX B - PLATE SETUP FOR THE Chromo4™ OR THE CFX96™

THERMOCYCLERS

APPENDIX C - PLATE SETUP FOR THE MiniOpticon™

THERMOCYCLER

1 - INTRODUCTION

Brettanomyces bruxellensis is a type of yeast that is responsible for the presence of 4-ethyl Phenol and 4-ethylguaiacol in wine-type drinks, fruit juices and beers that involves significant economic losses. In wines, its presence is difficult to detect using classic culture methods, which take a long time and are non-specific.

Rapid, specific and early detection of this yeast in the wine production would, however, allow the oenologist and the producer to take preventive measurements and to eliminate it before the appearance of the phenolated character that is characteristic of changes induced by this yeast. Compared with the traditional microbiological method, molecular biology provides high speed, high sensitivity and high specificity detection solutions.

Diagnosis of the presence of this yeast must make it possible to determine at what risk level the wine is and how this risk develops over time. This risk is variable according to the contamination level of the wine. Therefore, an adapted quantification tool is used to determine whether (i) the population is low and therefore that risk is low or even controlled, (ii) regular monitoring is required and the wine can be processed, in intermediate or critical quantities in the event that the wine must be supervised (iii) and finally whether the population is very high and in the event that the risk of producing volatile phenols is also high, an immediate and urgent action is required for the wine.

In all cases, regular monitoring is recommended. It is important to monitor the development Brettanomyces bruxellensis of in the wine.

VINEO™ Brettanomytest PCR Kit is a quantitative test used for specific detection of Brettanomyces bruxellensis in wines and grape musts in fermentation using the real-time polymerase chain technique (RT-PCR). A specific sequence of Brettanomyces bruxellensis is amplified and detected simultaneously by means of a fluorescent probe. Implementation of this test is used to obtain a quantitative result less than 3 hours after extracting the DNA (VINEO™ Extract DNA Kit, 354-8100). The analysis software adapted to these tests, Opticon Monitor™ or CFX Manager™ Industrial Diagnostic Edition, make it possible to measure the Brettanomyces bruxellensis risk in the analysed wine sample by an automatic and quantitative analysis. An interpretation of the risk level is proposed to the user in this way. It is linked to the number of CFU.ml-1 (Colony-forming unit) detected in the wine:

• 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.

This test is used to detect and quantify Brettanomyces bruxellensis in the DNA extracted from fermented drinks produced from grape and fermenting grape musts.

3 - KIT CONTENTS

This kit contains the quantity of reagent required for 96 reactions.

4 - VALIDITY AND PRESERVATION

On reception, the kit must be stored at temperatures between +2°C and +8°C. Each reagent stored between +2°C and +8°C can be used until the expiry date indicated on the tube.

NB: Do not freeze the reagents.

5 - REQUIRED EQUIPMENT THAT IS NOT PROVIDED

Equipment:

• Industrial Diagnostic CFX96™ real-time PCR detection system, 96 wells, ref. Bio-Rad :359-3990

• CFX Manager™ Software, Industrial Diagnostic Edition, ref. Bio-Rad : 359-3893

• MiniOpticon™ system for real-time PCR, reaction block of 48 wells, ref. Bio-Rad: 359-3995

• Vortex

• Optionally: centrifuge with rotor for plates or strips (max. 2000 x G)

• 20 µl, 200 µl and 1000 µl micropipettes

• Multi-distributor such as combitip pipettes

Note: We recommend using an uninterruptible power supply with the thermocycler.

Stoppers Designation Reagent Volume

Pink Fluorescent probes Fluorescent probes 1 tube (0.55 mL) White Amplification mix Amplification mix 2 tubes (2 x 2.2 mL) Green Negative PCR control Negative PCR control 1 tube (0.5 mL)

Red Positive Qs PCR control Positive Qs PCR control 1 tube (0.5 mL)

TAKING

THE SAMPLE

DNA EXTRACTION

VINEO™ Extract

DNA Kit

(Ref.: 354-8100)

DNA AMPLIFICATION by PCR IN REAL TIME

VINEO™

Brettanomytest PCR

Kit (Ref.: 354-8101)

ANALYSIS & INTERPRETATION Opticon Monitor™

CFX Manager™ IDE

Consumables:

• For the plates, PCR tubes and stop caps:

- White plates of 96 "Low-profile" wells, ref. Bio-Rad: MLL-9651

- White plates of 48 "Low-profile" wells, ref. Bio-Rad: MLL-4851

- Strips of 8 tubes of 200l "Low-profile", ref. Bio-Rad: TLS-0851

- Strips of 8 flat optical stop caps for tubes or plates of 0.2 mL wells, 120 strips ref. Bio-Rad: TCS-0803

• Cones for Combitip or multi-distributor, sterile when packaged

individually

• Cones with sterile filters that are adaptable to 10 µL, 20 µL, 100 µL,

200 µL and 1000 µL micro-pipettes

• 2mL and 5mL sterile tubes

• Non-powdered latex or nitrile gloves

• Sterile distilled water

• 5% Chlorine Bleach

• Decontaminating agent such as DNA AWAY

or RNAse AWAY

6 - PRECAUTIONS AND RECOMMENDATIONS

• This trial must be carried out by staff who have had adequate

training.

• Result quality depends on scrupulous compliance with Laboratory

Good Practice, in particular in the area of PCR:

- The equipment (pipettes, tubes etc.) must no go from one work station to another.

- It is indispensable to use a positive and a negative control for each series of amplification reactions.

- Do not use the reagents beyond their expiry date.

- Subject the reagents in the kit to a vortex movement using the apparatus provided for this purpose before using them to work with homogeneous solutions.

- Check the exactness and the precision of the pipettes as well as the proper functioning of the instruments.

- Change gloves regularly and as soon as you suspect that they may have been contaminated.

- Clean work surfaces regularly with a 5% solution of chlorine bleach and another agent such as DNA AWAY

or RNase AWAY®.

- Wear non-powdered gloves so as not to leave fingerprints on the optical film used to seal the micro-plates. Do not write on the PCR tube stoppers. In both cases, recording the data by the device can be disturbed.

We strongly recommend that you read the complete protocol before beginning the trial. Follow the suggested protocol scrupulously.

7 - SAMPLING AND TRANSPORT OF SAMPLES

Wine samples are collected under aseptic conditions in sterile glass or polyethylene recipients or recipients made out of similar material. The samples must be submitted to the laboratory as quickly as possible, preferably within 24 and not more than 48 after taking the sample. If the samples are transported and analysed within 24 h, transport and storage take place at ambient temperature (+18°C to +30°C). If the samples are transported and analysed within 48 h, transport and storage take place at between +2°C to +8°C.

8 - DNA EXTRACTION

DNA extraction must be carried out using the kit developed by Bio-Rad to extract DNA from wine sample: VINEO™ Extract DNA Kit (ref. Bio-Rad 354-8100).

9 - REAL-TIME PCR

1. Starting up the PCR apparatus

Switch the thermocyler and the computer on in that order and start up the Opticon Monitor software. For more information and for the definition of software parameters, see the user guide for the Bio-Rad thermocycler for the VINEO™ Brettanomytest PCR Kit.

2. Preparing PCR reactions

2.1 Prepare the VINEO™ Brettanomytest PCR Kit reagent mixture by mixing

(40 µL/sample) the amplification solution (tube with white stopper) and (5 µL/sample) the fluorescent probes (tube with pink stopper). Carry out the reagent mixture on the basis of the number of samples and controls to be analysed (at least 1 duplicate (2) of the positive Qs PCR control and a negative control must be used per plate). The PCR amplification reagent mixture preparation table in Appendix A indicates the required quantities of each reagent to be mixed on the basis of the number of samples to be analysed. Do not forget to include the 2 control points.

Note: do not mix reagent batches.

2.2 After preparation, the reagent mix (amplification and probe solutions)

must be used immediately, or can be stored for 2 hours between +2°C and +8°C.

2.3 Distribute 45 μl of this reagent mixture per well according to the defined plate surface. Appendices B and C propose plate surfaces to be used for the Chromo4™/CFX96™ and MiniOpticon™ thermocyclers respectively.

Add 5 μl of sample or negative control or Qs PCR positive control and seal the wells on the plate or the strips hermetically. It is important to insist on sealing the wells and the strips to avoid any evaporation phenomenon during the PCR reaction. It is important to avoid the presence of bubbles at the bottom of the wells by pipetting cautiously. To eliminate bubbles after sealing the plate or closing the PCR strips, you may centrifuge the plate of PCR strips briefly. The plate can be store at +2°C to +8°C for 2 hours.

2.4 Put the plate or the PCR strips into the thermocycler. Make sure that they are correctly oriented (well A1 at the top left). Close the reaction module.

3. Starting the amplification reaction

To restart the PCR, refer to the user guide for the Bio-Rad thermocycler for the VINEO™ Brettanomytest PCR Kit.

10 - ANALYSIS OF THE DATA

Data analysis can be carried out directly at the end of the amplification reaction or later by re-opening the data file. To open the data files and analyse the results of the PCR, refer to the user guide for the Bio-Rad thermocycler for the VINEO™ Brettanomytest PCR Kit.

11 - INTERPRETATION OF THE RESULTS

To obtain the analysis results, you only have to read the values of Ct (Cycle threshold): value of the amplification cycle from which fluorescence increases significantly above background noise.

Manual analysis will only be used for a qualitative analysis (presence or absence).

You must use the the Opticon Monitor™ software (version 3.3 and higher) or the CFX Manager™ software Industrial Diagnostic Edition for an automated and quantitative interpretation. A complete report may be printed (cf. software instructions).

1. Controls

Before a final interpretation of the results, you have to check the results of the negative and positive controls.

For the test to be valid, the results of the negative and positive controls must be as follows:

* N/A means “Not Applicable”. The software indicates N/A for the Ct of a sample when the fluorescence curve does not cross the threshold.

If the results of the positive and negative PCR Qs controls are different from those described in the table below, you have to begin the PCR again.

2. Samples

A sample is considered to be positive for Brettanomyces bruxellensis if the value of Ct ≥ 10 is obtained for the FAM fluorophore.

If no value is obtained for Ct

FAM

(Ct

FAM

= N/A), the interpretation of the result

depends on the value of the internal control:

- A sample is considered to be negative for Brettanomyces bruxellensis if

no value of Ct is obtained for the FAM fluorophore (Ct

FAM

= N/A and if the

Ct of the internal control is greater than or equal to 28. (Ct

HEX

≥ 28).

- An N/A value for the Ct

HEX

of the internal control indicates that an

inhibition phenomenon of the PCR reaction has probably occurred, if the Ct

FAM

of the target is also N/A. In this case, the DNA sample must

be diluted (to 1/10

for example) in sterile distilled water and then

subjected to a new PCR.

- If the Ct

HEX

of the internal control is less than 28, it is impossible to

interpret the result. Check that the threshold was set correctly or that the raw curve shows an aspect characteristic of exponential amplification. If the observed curve is not correct, it will be necessary to repeat the PCR test for this sample.

Detection of Brettanomyces

bruxellensis (FAM)

Detection of the internal

control (Channel HEX)

Negative control Ct = N/A* 25 ≤ Ct ≤ 40

Positive Qs PCR control 27 ≤ Ct ≤ 37 Not significant

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 1/5thor 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 l 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.

Detection of Brettanomyces

bruxellensis (FAM)

Detection of the internal

control

Interpretation

Ct ≥ 10*** Not significant Positive

Ct = N/A

HEX

≥ 28

Negative

Ct = N/A Ct = N/A Inhibition**

APPENDIX A - PCR AMPLIFICATION REAGENT MIXTURE PREPARATION TABLE

Use this table to determine the required quantities of fluorescent probes and amplification solution to prepare the PCR reagent mixture. Calculation of the number of samples

must include the 2 positive Qs PCR controls and the negative control.

Number

of samples

Fluorescent probes (μL)

(Pink stopper)

Ampliﬁcation

solution (μL)

(White stopper)

Number

of samples

Fluorescent

probes (μL)

(Pink stopper)

Ampliﬁcation

solution (μL)

(White stopper)

1 5 40 49 265 2117 2 11 86 50 270 2160 3 16 130 51 275 2203 4 22 173 52 281 2246 5 27 216 53 286 2290 6 32 259 54 292 2333 7 38 302 55 297 2376 8 43 346 56 302 2419

9 49 389 57 308 2462 10 54 432 58 313 2506 11 59 475 59 319 2549 12 65 518 60 324 2592 13 70 562 61 329 2635 14 76 605 62 335 2678 15 81 648 63 340 2722 16 86 691 64 346 2765 17 92 734 65 351 2808 18 97 778 66 356 2851 19 103 821 67 362 2894 20 108 864 68 367 2938 21 113 907 69 373 2981 22 119 950 70 378 3024 23 124 994 71 383 3067 24 130 1037 72 389 3110 25 135 1080 73 394 3154 26 140 1123 74 400 3197 27 146 1166 75 405 3240 28 151 1210 76 410 3283 29 157 1253 77 416 3326 30 162 1296 78 421 3370 31 167 1339 79 427 3413 32 173 1382 80 432 3456 33 178 1426 81 437 3499 34 184 1469 82 443 3542 35 189 1512 83 448 3586 36 194 1555 84 454 3629 37 200 1598 85 459 3672 38 205 1642 86 464 3715 39 211 1685 87 470 3758 40 216 1728 88 475 3802 41 221 1771 89 481 3845 42 227 1814 90 486 3888 43 232 1858 91 491 3931 44 238 1901 92 497 3974 45 243 1944 93 502 4018 46 248 1987 94 508 4061 47 254 2030 95 513 4104 48 259 2074 96 518 4147

APPENDIX B PLATE SETUP FOR THE Chromo4™/CFX96™ THERMOCYCLERS

The first 3 wells in column 1 may be used to analyse the 3 control points. The other wells in the plate will be used for the samples to be analysed.

NC: Negative PCR control QS: Positive Qs PCR control

APPENDIX C PLATE SETUP FOR THE MiniOpticon™ THERMOCYCLER

The first 3 wells in column 1 may be used to analyse the 3 control points. The other wells in the plate will be used for the samples to be analysed.

NC: Negative PCR control QS: Positive Qs PCR control

1 2 3 4 5 6 7 8 9 10 11 12 A QS B QS C NC D Sample E Sample F … G H

1 2 3 4 5 6 A QS B QS C NC D Sample E Sample F … G H

NOTICE OF CONCERN TO THE PURCHASER: LIMITED LICENSE

Use of this product is covered by one or more of the following US patents and the corresponding patent claims outside the United States: 5 079 352, 5 789 224, 5 618 711, 6 127 155, 5 677 152 (claims 1 to 23 only) and 5 773 258 (claims 1 and 6 only) and by the claims outside the United States that correspond to US patent n° 4 889 818. The purchase of this product includes limited immunity from legal proceedings that may not be transferred according to the claims of previous patents when this quantity of product is solely used for alimentary analysis, environmental and industrial microbiology purposes including publication of the results of the purchaser's activities in return for payment or other commercial counterpart, and when it is also used for the purchaser's own research purposes. No right according to any patent claim (such as the claims of method 5'-nuclease in US patents n° 5 210 015 and 5 487 972) is not expressly transferred whether by implication or preclusion. Further information on the purchase of licenses may be obtained by contacting the Director of Licenses, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, United States.

VINEO™Brettanomytest PCR Kit

Réf. : 354-8101

Notice d’utilisation

Test pour la détection et quantification par PCR en temps réel de Brettanomyces bruxellensis

SOMMAIRE

1 - INTRODUCTION

2 - PRINCIPE DU VINEO™ Brettanomytest PCR Kit

3 - COMPOSITION DU KIT

4 - VALIDITE ET CONSERVATION

5 - EQUIPEMENT ET MATERIEL NECESSAIRES NON FOURNIS

6 - PRECAUTIONS ET RECOMMANDATIONS

7 - PRELEVEMENT ET TRANSPORT DES ECHANTILLONS

8 - EXTRACTION DE L’ADN

9 - PCR EN TEMPS REEL

10 - ANALYSE DES DONNEES

11 - INTERPRETATION DES RESULTATS

12 - PROTOCOLE DE CONFIRMATION A PARTIR DE COLONIES ISOLEES

13 - PERFORMANCES DU TEST ET VALIDATIONS

ANNEXE A - TABLEAU DE PREPARATION DU MELANGE

REACTIONNEL D’AMPLIFICATION PCR

ANNEXE B - PLAN DE PLAQUE A UTILISER SUR LES

THERMOCYCLEURS Chromo4™ OU CFX96™

ANNEXE C - PLAN DE PLAQUE A UTILISER SUR LE

THERMOCYCLEUR MiniOpticon™

1 - INTRODUCTION

Brettanomyces bruxellensis est une levure responsable de la présence des 4-ethyl Phénol et 4-ethyl Guaiacol dans les boissons de type vins, jus de fruits et bières impliquant des pertes économiques importantes. Dans les vins, sa présence est difficile à détecter par les méthodes de culture classiques qui sont longues et non spécifiques.

La détection rapide, spécifique et précoce de cette levure dans le processus d’élaboration des vins permettrait cependant à l’œnologue et au producteur de prendre des mesures de prévention et de l’éliminer avant l’apparition du caractère phénolé, caractéristique de l’altération par cette levure. Face à la méthode microbiologique traditionnelle, la biologie moléculaire apporte des solutions de détection à de hauts niveaux de rapidité, de sensibilité et de spécificité.

Le diagnostic de la présence de cette levure doit permettre de déterminer à quel niveau de risque se trouve le vin et comment évolue ce risque dans le temps. Ce risque est variable selon le niveau de contamination du vin. Ainsi un outil de quantification adapté permet de déterminer si (i) la population est en faible quantité et donc que le risque est faible voire maîtrisé, (ii) en quantité intermédiaire ou critique, dans ce cas le vin doit être surveillé, un suivi régulier s’impose, le vin peut être traité (iii) et enfin si la population est très importante, et dans ce cas le risque de production des phénols volatils l’est aussi, une action immédiate et urgente s’impose sur le vin.

Dans tous les cas un suivi régulier est préconisé. Il est important de suivre l’évolution des Brettanomyces bruxellensis au sein du vin.

VINEO™ Brettanomytest PCR Kit est un test quantitatif permettant la détection spécifique de Brettanomyces bruxellensis dans les vins et les moûts en fermentation par la technique de polymérisation en chaîne en temps réel (RT-PCR). Une séquence d’ADN spécifique de Brettanomyces bruxellensis est amplifiée et détectée simultanément grâce à une sonde fluorescente. La mise en œuvre de ce test permet l'obtention d'un résultat quantitatif moins de 3h après l’extraction d’ADN (VINEO™ Extract DNA Kit, 354-8100). Les logiciels d’analyse adaptés à ce test, Opticon Monitor™ ou CFX Manager™ Industrial Diagnostic Edition, permettent par une analyse automatique et quantitative, de mesurer le risque Brettanomyces bruxellensis dans l’échantillon de vin analysé. Une interprétation du niveau de risque est ainsi proposée à l’utilisateur. Elle est liée au nombre d’UFC.mL-1 (Unité Formant Colonie) détecté dans le vin :

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