hygiena KIT230140 (LP) Spoilage Yeast Detection 3 LyoKit Instructions

hygiena KIT230140 (LP) Spoilage Yeast Detection 3 LyoKit Instructions

What this Product Does

Number of Tests

The kit is designed for 48 reactions with a final reaction volume of 25 µL each. Up to 46 samples (single
sample preparation) plus positive control and negative control reactions can be analyzed per run.

Storage and Stability

• Store kit at 2 to 8 °C through expiration date printed on label.
• Once kit is opened, store components as described in the following Kit Contents table:

Kit Contents

Component	Description	Contents / Function / Storage
foodproof® Spoilage Yeast Detection 3 LyoKit Microplate, prefilled with 48 reactions (lyophilized)	Aluminum bag containing an 8-tube strip mat KIT230140 with white low-profile (LP) tubes KIT230141 with clear regular-profile (RP) tubes KIT230142 with clear deep-profile (DP) tubes	48 refilled reactions (philosophized). Ready-to-use PCR mix containing primer and hydrolysis probes specific for DNA of designated spoilage yeasts and Internal Control (IC), as well as, Taq DNA Polymer and Uracil-DNA N- Glycogen (UNG, heat labile) which is for prevention of carry- over contamination. Store at 2 to 8 °C in supplied aluminum bag with the silica pad (Keep tightly sealed) Protect from light and moisture!
Control Template	Vial 2 (purple cap)	1 x 350 µL Contains a stabilized solution of DNA. For use as a PCR run positive control. Store at 2 to 8 °C.
H2O PCR-grade	Vial 3 (colorless cap)	1 x 1 mL Nuclease-free, PCR-grade H2O. For use as a PCR run negative control.
Cap strips	Plastic bag containing 8- cap strips	12 x 8-cap strip For use in real-time PCR after addition of samples.

Additional Equipment and Reagents Required

• Real-time PCR cycler suitable for detection of FAM, HEX, ROX and Cy5/ATTO 490LS-labeled probes. For situations where PCR strip tubes don´t fit the cycler, samples can be transferred to appropriate PCR vessels after re suspension of the philosophized PCR mix. Take precautions to avoid cross contamination during this pi petting step.
• Sample Preparation Kit foolproof Star Prep Two Kit (Product No. KIT230177)
• Reagent D (Product No. KIT230001)
• Nucleate-free, aerosol-resistant pipette tips
• Pipettes
• Vortex centrifuge Multi spin MSC-6000 for PCR strips with SR-32, Rotor for MSC-3000/6000 or Vortex centrifuge CVP-2 for PCR plates

Applicability Statement

The foolproof Spoilage Yeast Detection 3 Toolkit is intended for rapid qualitative detection of spoilage yeast DNA isolated from a wide range of beverage samples that are potentially contaminated with Saccharomyces cerevisiae var. statisticians and/or Remittances/Baedeker app. DNA from dead yeast can be excluded from analysis by use  of Reagent D and a suitable protocol.

The kit must not be used in diagnostic procedures.

The kit described in these Product Instructions has been developed for real-time PCR instruments.

• Versions KIT230140 and KIT230141 are designed for instruments with FAM, VIC/HEX, ROX and Cy5 detection channels. The performance of the kit was tested with the following real-time PCR instruments: LightCycler® 480, LightCycler 96 (Roche Diagnostics), AriaMx® and Mx3005P® (Agilent Technologies), ABI™ 7500 Fast (Thermo Scientific), QuantStudio™ 5 (Thermo Scientific) and CFX96™ (Bio-Rad).
• Version KIT230142 is designed for instruments with FAM, VIC/HEX, ROX and ATTO 490LS detection channels.
  The performance of the kit was tested with the Dualo 32® Beverage PCR instrument (Hygiena Diagnostics).

Note: Color Compensation is necessary and will be supplied by Hygiena Diagnostics for users of the LightCycler 480 System I and LightCycler 480 System II (Color Compensation Set 5; Product No. KIT230011).

How to Use this Product

Before You Begin

Precautions

Detection of spoilage yeast DNA using the foodproof Spoilage Yeast Detection 3 LyoKit requires DNA amplification by PCR. The kit provides all reagents required for the PCR. However, in order to achieve reliable results, the entire assay procedure must be performed under nucleate-free conditions. Follow the instructions below to avoid nuclease-, carry-over-, or cross-contamination:

• Keep the kit components separate from other reagents in the laboratory.
• Use nuclease-free lab ware (e.g., pipettes, pipette tips, reaction vials).
• Wear gloves when performing the assay.
• To avoid cross-contamination of samples and reagents, use fresh aerosol-preventive pipette tips.
• To avoid carry-over contamination, transfer the required solutions for one experiment into a fresh tube, rather than directly pipetting from stock solutions.
• Physically separate the workplaces for DNA preparation, PCR setup, and PCR to minimize the risk of carry-over contamination. Use a PCR hood for all pipetting steps.

Keep the foodproof Spoilage Yeast Detection 3 LyoKit philosophized PCR Mix away from light and moisture!

Sample Material
Use any sample material suitable for PCR in terms of purity, concentration, and absence of inhibitors. For
preparation of genomic DNA from various samples, refer to the corresponding product package inserts of a
suitable sample preparation kit (see “Additional Equipment and Reagents Required”).

DNA Extraction
Hygiena Diagnostics provides sample preparation kits suitable for all kinds of food and environmental samples
(see “Additional Equipment and Reagents Required”). For more product information, please refer to
www.hygiena.com

Positive Control
Always run a positive control with the samples. To prepare a positive control, replace the template DNA with the
provided control DNA [foolproof Spoilage Yeast Detection 3 Control Template (vial 2, purple cap)] or with a positive sample preparation control.

Negative Control
Always run a negative control with the samples. To prepare a negative control, replace the template DNA with
foodproof Spoilage Yeast Detection 3, PCR-grade water (vial 3, colorless cap). Include a negative control during
sample preparation to monitor reaction purity and cross-contamination. This extraction control can be used as an additional negative control reaction.

Procedure

Program Setup for the Dualo® 32 Beverage Instrument (KIT230142)
The Dualo 32® Beverage (Product No. MCH230008) can be started from a pre-installed run template: Click on “New”, select the appropriate template, and press “Select”. After loading the samples, the instrument can be started by clicking on “Start Run”.

For detailed instructions on how to program and start the PCR run on the Dualo 32® Beverage, please refer to
the manual for this instrument.

Program Setup for other cyclers (KIT230140 / KIT230141)
The following procedure is optimized for real-time PCR instruments with FAM (Saccharomyces cerevisiae var. statistician s), VIC/HEX (Remittances/Baedeker app.), ROX (B. Brunelleschi and B. anomalus) and Cy5 or ATTO 490LS (Internal Control) detection channels. Program the PCR instrument before preparing the PCR samples. For details on how to program the experimental protocol, see the Instrument Operator’s Manual for the real-time PCR cycler used.

Use the following real-time PCR protocol for the foodproof Spoilage Yeast Detection 3 LyoKit:

Pre-incubation: 1 cycle

Step 1: 37 °C for 4 minutes
Step 2: 95 °C for 10 minutes

Amplification 50 cycles*
Step 1: 95 °C for 5 seconds
Step 2**: 60 °C for 60 seconds

* Minimum denaturation time may be longer on some instruments. Set to the shortest denaturation time possible in this case.
** Fluorescence detection in Step 2

Notes:

• For some real-time PCR instruments, the type of the probe quencher as well as the use of a passive reference dye has to be specified. The foolproof Spoilage Yeast Detection 3 LyoKit contains probes with a non-fluorescent (“dark”) quencher and no passive reference dye.

Preparation of the PCR Mix

Proceed as described below to prepare a 25 µL standard reaction. Always wear gloves when handling strips or caps.
Use any sample material suitable for PCR in terms of purity, concentration, and absence of inhibitors.

Note: The PCR strips must be stored in the provided aluminum bag with the silica gel pads to avoid liquid absorption.

Qualitative Detection

1. Take the needed number of PCR tube strips out of the aluminum bag. Use scissors or a scalpel to cut the strips apart. Tightly seal the bag afterwards and store under the recommended conditions.
2. Place the PCR tube strips containing the lyophilized reagents in a suitable PCR tube rack. Check that the reagent pellets are at the bottom of the tubes. If not, briefly centrifuge or flick the pellets to the bottom before proceeding.
3. Uncap the tube strips cautiously and discard the cap strips.
   Note: Do not leave strips open for extended periods of time. To avoid unwanted liquid absorption, open strips only shortly before filling.
4. Pipette 25 µL sample into each PCR vessel:
   • For the samples of interest, add 25 µL sample DNA (if using less volume, add PCR-grade H2O to achieve 25 µL).
   • For the negative control, add 25 µL PCR-grade H2O (vial 3, colorless cap).
   • For the positive control, add 25 µL foodproof Spoilage Yeast Detection 3 Control Template (vial 2, purple cap)
     Note: To reduce the risk of cross-contamination, it is recommended to prepare only one PCR tube strip at a time.
5. Seal the vessels accurately and tightly with the colorless cap strips.
6. Mix thoroughly using a vortex centrifuge.
   Note: Hygiena Diagnostics recommends vortex centrifuge Multispin MSC-6000 for PCR strips or vortex centrifuge CVP-2 for PCR plates. Dedicated protocols are available for these centrifuges upon request.
   Note: Alternatively resuspend the pellet by manual mixing. This may be achieved by cautiously pipetting the sample up and down multiple times during step 4 or flipping the tube strips after sealing while  pressing down the cap strip.
7. Spin the PCR tube strips for 30 seconds at 150 – 200 x g in a suitable centrifuge.
   Note: If your centrifuge exceeds 200 x g, do not centrifuge for more than 5 seconds. Avoid centrifugation forces exceeding 1000 x g!
8. Place the samples in your PCR cycler and run the program as described above.
   Note: When using any LightCycler 480 instrument, a special adapter (Product No. MIS230005) is necessary.
   For some PCR instruments, the PCR strips should be placed in a balanced order into the cycler block. For example, two strips can be placed in columns 1 and 12.

Data Interpretation

Amplification of DNA specific for Saccharomyces cerevisiae var. statisticians is analyzed in the fluorescence channel suitable for FAM-labeled probes detection. DNA-specific amplification for Remittances/Baedeker app. is analyzed in the fluorescence channel suitable for VIC/HEX. Amplification of DNA specific for Remittances
Brunelleschi and Remittances anomalous is analyzed in the fluorescence channel suitable for ROX. Specific amplification of the Internal Control is analyzed in the fluorescence channel suitable for Cy5/ATTO 490LS.

Compare the results from channel FAM, VIC/HEX, ROX and channel Cy5/ATTO 490LS (Internal Control) for each sample, and interpret the results as described in the table below.

Qualitative Detection

For qualitative detection, compare the results from channels FAM, VIC/HEX, ROX and channel Cy5/ATTO 490LS (Internal Control) for each sample and interpret the results as described in the table below:

Channel FAM	Channel HEX	Channel ROX	Channel Cy5/ATTO 490LS	Result Interpretation
Positive	Positive or Negative	Positive or Negative	Positive or Negative	Positive for Saccharomyces cerevisiae var. statisticians
Positive or Negative	Positive	Positive or Negative	Positive or Negative	Positive for Remittances/Baedeker spp.
Positive or Negative	Positive	Positive	Positive or Negative	Positive for B. Brunelleschi and/orB. anomalous
Negative	Negative	Negative	Positive	Negative for targeted spoilage yeasts
Negative	Negative	Negative	Negative	Invalid

Troubleshooting

Observation	Possible Reason	Recommendation
No signal increase is observed, even with positive controls.	Incorrect detection channel has been chosen.	Set Channel settings to FAM, HEX, ROX and Cy5 or ATTO 490LS. If your instrument does not have a HEX Channel, use VIC instead.
	Pipetting errors.	Check for correct reaction setup. Repeat the PCR run. Always run a positive control along with your samples.
	No data acquisition programmed.	Check the cycle programs.
No signal increase in channel Cy5 is observed, with other channels also negative.	Inhibitory effects of the sample material (e.g., caused by insufficient purification).	Use the recommended DNA sample preparation kit to purify template DNA. Dilute samples or pipette a lower amount of sample DNA (e.g., 20 µL PCR-grade H2O and 5 µL sample DNA instead of 25 µl sample DNA).
Fluorescence intensity is too low.	Inappropriate storage of kit components.	Store the foolproof Spoilage Yeast Detection 3 LyoKit philosophized PCR Mix at 2 to 8 °C, protected from light and moisture.
	Low initial amount of target DNA.	Increase the amount of sample DNA Depending on the chosen DNA isolation method, inhibitory effects may occur.
Strong decrease of fluorescence baseline	Re suspension of philosophized PCR mix not complete	Always re suspend philosophized PCR mix thoroughly.
Negative control samples are positive.	Carry-over contamination.	Exchange all critical solutions. Repeat the complete experiment with fresh aliquots of all reagents. Always handle samples, kit components and consumables in accordance with commonly accepted practices to prevent carry- over contamination. Add positive controls after sample and negative control reaction vessels have been sealed.
Fluorescence intensity varies.	Insufficient centrifugal of the PCR strips. Re suspend PCR mix is still in the upper part of the vessel.	Always centrifuge PCR strips. Check that no air bubbles are formed or remain in tube after centrifugal.
	Outer surface of the vessel or the seal is dirty (e.g., by direct skin contact).	Always wear gloves when handling the vessels and seal.
Pellets are difficult to dissolve.	The lyophilized PCR mix started to rehydrate.	Store the philosophized PCR mix always in the aluminum bag with the silica gel pad. Open strip shortly before filling.

Additional Information on this Product

How this Product Works
The foodproof Spoilage Yeast Detection 3 LyoKit provides all necessary reagents and a control template for
reliable interpretations of results. To ensure maximum reliability of the kit and to prevent misinterpretation of
negative results due to inhibition of the amplification, an Internal Control (IC) is included. A hydrolysis probe was designed to bind specifically the IC, allowing detection in the Cy5 channel or ATTO 490LS channel, whereas the DNA from spoilage yeasts is detected in channels FAM, HEX and ROX. In case of a negative result due to inhibition of the amplification by the sample DNA extract of interest, the amplification of the IC is suppressed as well, whereas a negative result for the sample DNA extract of interest and amplification of the IC clearly indicates the absence of spoilage yeast DNA in the sample. The foodproof Spoilage Yeast Detection 3 LyoKit minimizes contamination risk and contains all reagents (except for template DNA) needed for the detection of spoilage yeast DNA. Primers and probes provide specific detection of spoilage yeast DNA in food and beverage samples. The described performance of the kit is guaranteed for use on the real-time PCR instruments listed above only.

Test Principle

1. Using the kit’s sequence-specific primers in a polymer chain reaction (PCR), the PCR instrument and the supplied reagents amplify fragments of genomic DNA originating from spoilage yeasts belonging to the species Saccharomyces cerevisiae var. statisticians and Remittances/Baedeker spp.
2. The PCR instrument detects these amplified fragments in real time through fluorescence generated by cleavage of the hybridized probe due to the 5´-nucleate activity of the Taq DNA polymer. The probe is labeled at the 5´-end with a reporter fluoroscope and at the 3´-end with a quencher.
3. During the annealing/elongation phase of each PCR cycle, the probe hybridizes to an internal amplification sequence and is cleaved by the 5’ nuclease activity of the Taq DNA polymer. This cleavage of the probe separates the reporter dye from the quencher dye, increasing the reporter dye signal.
4. The PCR instrument measures the emitted fluorescence of the reporter dye.

Prevention of Carry-Over Contamination

The heat-labile Uracil-DNA N-Glycosylase (UNG) is suitable for preventing carry-over contamination between PCRs.
This technique relies on the incorporation of deoxyuridine triphosphate (dUTP) during all amplification reactions and the pretreatment of all successive PCR mixtures with the heat-labile UNG. The UNG cleaves DNA at any site where a deoxyuridine residue has been incorporated. The resulting abasic sites are hydrolyzed due to the high temperatures during the initial denaturation step and can no longer serve as PCR templates. The heat-labile UNG is inactivated during the initial denaturation step. Native DNA (e.g., the isolated yeast genomic DNA) does not contain uracil and is therefore not degraded by this procedure. Since dTTP is replaced with dUTP and UNG is included in the foodproof Spoilage Yeast Detection 3 LyoKit, decontamination can be achieved with the provided reagents.

Background Information

Spoilage yeasts are usually defined as species or strains of yeast unintentionally introduced into a fermentation
process or final product, which can compromise the quality of food and beverages. Extreme examples of yeast
spoilage include exploding packages, “blown cans” of soft drinks, cloudy re-fermented wine, pink or red slime dripping from refrigerated meat, white yeast colonies on food, and tainted fruit juices [1].

Saccharomyces cerevisiae var. statisticians is considered one of the most dangerous spoilers of beer, as it ferments extrinsic, leading to super-attenuation. This yeast is particularly noted for its ability to extracellular secrete amylase, which is encoded by the STA1 gene, and to ferment starch [2,3].

Saccharomyces cerevisiae var. statisticians causes extremely high bottle internal pressures, leading even to
explosions and sensory changes such as dry mouthfeel and phenol off-flavor [4]. The genus Baedeker/Remittances is comprised of five recognized species: B. anomalous, B. Brunelleschi, B. Austronesian, B. casehardens and B. anus. Baedeker/Remittances yeast are spoilers of many beverages, such as beer, juice and wine, and have harmful effects on flavor and/or visual appearance. These yeasts produce acetic acid, 4-ethyl phenol and 4-ethylguaiacol, causing off-flavors in wine. Baedeker/Remittances species also cause haze and turbidity in bottled wine and iambic beer [5]. It is possible that resistance to citric acid, together with the ability to utilize nitrate, may enhance the ability of Baedeker/Remittances spp. to spoil low-nutrient soft drinks
[1]. Therefore, for beverage manufacturers, control of these species is very important to prevent spoilage
incidents in their products. Generally, Baedeker/Remittances yeasts grow slowly on culture media, and the
detection of spoilage Baedeker/Remittances strains typically takes 3–7 days, depending on the medium used for
detection. Therefore, rapid detection and identification methods for Baedeker/Remittances yeasts have been previously reported. For example, the polymer chain reaction (PCR) method [5]. Baedeker/Remittances spp. present markedly different ability in their potential to spoil different beverages. B. anomalous and B. Brunelleschi present the largest potential, being able to spoil even low pH, high alcohol beverages such as beer and wine. Also, B. Austronesian exhibits beer spoilage properties [5]. In contrast, the growth of B. anus and B. casehardens appears to be limited in beer but these yeasts have been reported as important spoilers of beer mixed beverages and non-alcoholic beverages [1,2,5,6].

References

1. Bertram, J., Stafford, M. (2006). Food and beverage spoilage yeasts. In: Querol, A.; Fleet, G.H.
   (eds.). The Yeast handbook volume 2: Yeasts in food and beverages. Sp ringer-Verlag, Berlin,
   Germany, p.336-379.
2. Hutzler M. (2009). Dissertation: Entwine ind Optimization con Method en our Identification und
   Differentiated von irrelevant Helen.
3. Yamagata I., Suzuki K., Fukuoka S. (1985). Nucleotide Sequence of the Extracellular Amylase Gene
   STAI in the Yeast Saccharomyces statisticians. J. Bacteriol. Microbiol., vol. 161, no. 2, p.567–573.
4. Hustler, M., Honeywell, U., Tense, Chr., Geiger, E. (2008). Beer mixed beverages: dangerous
   spoilage yeasts, susceptible beverages? Bracelet International IV p.206-211.
5. Shiatsu S, Casanova S, Imam K, Suzuki K, Maharishi H, Await M (2015) Investigation of beer-spoilage
   ability of Baedeker/Remittances yeasts and development of multiplex PCR method for beerspoilage yeasts. Journal of The Institute of Brewing. 121, 177-180.
6. Hustler, M., Riedl, R., Koob J., Jacob, FA. (2012). Fermentation and Spoilage Yeasts and their Relevance for the Beverage Industry – A Review. Brewing Science. 65, 33-52.

Product characteristics

The foolproof Spoilage Yeast Detection 3 LyoKit has been designed to detect all strains belonging to the species Saccharomyces cerevisiae var. statisticians and the genus Baedeker/Remittances by qualitative PCR. Performance has been tested with representative beverage matrices, e.g. beer, wine, non-alcoholic beverages and juices.

Specificity: The foolproof Spoilage Yeast Detection 3 LyoKit exclusivity has been tested with 45 strains including 23 strains of Saccharomyces cerevisiae var. statisticians and 22 strains of Baedeker/Remittances. The exclusivity was determined using 42 unrelated wild yeast species. No false positives or false negatives were determined.

Sensitivity: At least 102 cfu/ml can be directly detected from beverages and enrichment cultures with a sensitive protocol using the foodproof StarPrep Two Kit (Product No. KIT230177). A sensitivity of 1 cell per sampled volume can be achieved with suitable pre-enrichment procedures.

Quality Control
The foodproof Spoilage Yeast Detection 3 LyoKit is function tested using the LightCycler 480 System and the
Dualo 32 Beverage.

Supplementary Information

Ordering Information
Hygiena Diagnostics offers a broad range of reagents and services. For a complete overview and for more information, please visit our website at www.hygiena.com.

License Notice
The purchase price of this product includes limited, nontransferable rights under US Patent No. 7,687,247 owned by Life Technologies Corporation to use only this amount of the product to practice the claims in said patent solely for activities of the purchaser for bioburden testing, environmental testing, food testing, or testing for genetically modified organisms (GMO) in accordance with the instructions for use accompanying this product. No other rights are conveyed, including no right to use this product for in vitro diagnostic, therapeutic, or prophylactic purposes. Further information on purchasing licenses under the above patent may be obtained by contacting the Licensing Department, Life Technologies Corporation, 5791 Van Allen Way, Carlsbad, CA 92008.

Email: [email protected].

Trademarks
foodproof®, microproof®, vetproof®, ShortPrep®, StarPrep®, RoboPrep® and LyoKit® are registered trademarks of Hygiena Diagnostics GmbH. Hygiena® is a registered trademark of Hygiena. Other brand or product names are trademarks of their respective holders.

Contact and Support
If you have questions or experience problems with this or any other product of Hygiena Diagnostics GmbH, please contact our Technical Support staff (www.hygiena.com/support). Our scientists commit themselves to providing rapid and effective help. We also want you to contact us if you have suggestions for enhancing our product performance or using our products in new or specialized ways. Such customer information has repeatedly proven invaluable to us and the worldwide research community.

Reference Number
The reference number and original Hygiena Diagnostics GmbH article numbers:
R 602 69-1, R 602 69-2, R 602 69-3

Change Index

Version 1, July 2021
First version of the package insert.

Revision A, January 2024:
Rebranding and new layout.
R 602 69 20 -> INS-KIT230140-41-42-RevA

Hygiena®
Camarillo, CA 93012

USA
diagnostics.[email protected]

Manufactured by Hygiene Diagnostics GmbH
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14473 Potsdam
Germany
www.hygiena.com