Real-time PCR beer screening

Transcription

1 Real-time PCR beer screening For a quick and GEN-IAL result Avoid product recalls Minimize production losses Prevent damage to brand image For in-process control and/or for online final product control R-Biopharm for the safety of your analysis.

2 1 Potential sources of primary infection water malt bitter hops 2 yeast propagation/ storage optional aroma hops mash lautering wort whirlpool cooling fermentation storage Potential sources of secondary infection keg bottle filling storage flash pasteur filtration Brew water: e.g. Enterobacteriaceae Yeast slurry: contamination with e.g. Lactobacillus, non cultured yeast After filtration: main sources for detection of different beer spoilers Filling station: e.g. S. diastaticus Product: final quality product control, bacteria /yeast Rapid and reliable detection of beer spoilage bacteria and yeasts Due to low ph, alcohol content, hop, and fermentation of nutrition by the cultured yeast, beer is not a likely habitat for microbes. However, specialized bacteria and yeasts may adapt to this environment causing turbidity, change of taste and off-flavor. Also, secondary fermentation of S. diastaticus may pose a risk to consumers. To avoid spoiled brews and in the worst cases expensive recalls, breweries are testing, to different extents, for spoilage bacteria and yeasts in different places in the brewery. Traditionally, this is performed by filtration and incubation in dedicated media (anaerobic/aerobic) or broth for several days (ranging from 3-5 days up to 10 to 14 days depending on the organism) and either plaques on plates or cells under the microscope, will be identified visually. While this method has been used for several decades, and carries a low consumable cost, it is also time consuming and susceptible to misinterpretation, especially when mixed contaminations are involved. One may differentiate between spoilage (obligate) microorganisms (e.g. fast growing L. brevis, P.damnosus, L. backii, slow growing L. lindneri), potential spoilage germs (e.g. P. claussenii, L. buchneri) and non-spoilage germs which are indicators of poor hygiene (e.g. L. fermentum). Additionally the knowledge about presence/absence of hop resistance Yeast cells detected by microscope. Correct identification of bacteria and yeast cells especially in mixed contamination might be difficult.

3 genes may support a risk management. The exact and rapid detection and identification of species, as well as the source of contamination, will help the brew master take rapid action. Modern real-time PCR methods provide a variety of tools, while the procedure of this rapid detection (< 2 hours) is easy to use. The DNA based method gives highly specific information about the group and species of microbes. GEN-IAL GmbH presents the culmination of 20 years experience with the new QuickGEN line: Sampling and sample preparation: Depending on the current need, the sampling may be performed at different places: For end product control in bottles or kegs and at different stages during production, starting with the brewing water (Enterobacteriaceae), the yeast propagation tank (Lactobacillus and wild yeast) as well as during post fermentation steps. The sampling devices from R. Schütt allow for easy in-process sampling. The DNA based real-time methods allow for precise and specific detection and are based on: 1. DNA preparation and 2. detection. Different detection assays may be used with the DNA, e.g. for a combination of screening and detection. Preincubation: (CSOW 0100) The sample is preincubated in media/broth of choice (e.g. MRS, NBB, etc.) for 48 hours and 1 ml (or a colony) is used for lysis. Lysis buffer alone (CSEOW 0100) may be used if washing is not necessary (no dark beer). Centrifugation: (CSE 0100) 30 ml of beer can be centrifuged (in two steps to separate yeast from bacteria), then washed and used for lysis directly. This allows for rapid identification, however the sensitivity will be limited due to the centrifugation volume without pre-enrichment ( cfu / 30 ml). Filtration: (FSE 0100) Beer is filtered according to the standard procedure. Lysis is performed via a polycarbonate filter following the washing step. The sensitivity is dependent on the volume of beer which can be filtered without blocking (usually ml). PolyBIND : For highest sensitivity, beer can be analyzed online in a pipe or as product in a bottle/ keg using a polymer which binds the bacteria/yeast without blocking. Sampling is possible for up to 30 liters, either by online sampling or from bottles. After binding to the PolyBIND, the sample will be incubated anaerobically in media and analyzed like a preincubated sample. For most rapid analysis, 30 ml of beer will be centrifuged at g for 20 min and the pellet be used for analysis. Filtration is the most common sampling procedure. Sensitivity depends on the volume which can be filtrated without blocking. The most advanced and sensitive binding of microbes on PolyBIND requires specific sampling equipment (bottles/kegs or online) and is dedicated for midsize to large breweries.

4 Detection: For cost reasons and for different applications, different combinations of screening and identification are available 1. Group Specific Screening An initial screening gives a group specific answer. The multiplex screening requires a 4-plex real-time PCR block thermocycler (detecting fluorescent dyes FAM, HEX, ROX, Cy5). Examples include: MyGo Pro, Agilent MX3005P, Biorad CFX 96, ABI 7500, Roche Lightcycler 96, LC 480 and others. The costs for these machines are declining. Data extraction using amplification curves and Ct values for the channels can be easily performed and documented. QPP1T 0050 This assay may be used for post filtrated beer samples where no yeast should be present Lactobacillus spp. and Pediococcus spp. Megasphaera and Pectinatus Yeasts Internal Control QPP1HR 0050 This assay detects bacteria and provides additional risk assessment for hop resistance Lactobacillus spp. and Pediococcus sp. Megashpaera and Pectinatus Hop resistance genes hora/horc Internal Control QPP1H 0050 For use with the Lightcycler 2.0, the groups can be differentiated Lactobacillus spp. versus Pediococcus spp. Megasphaera versus Pectinatus Yeasts with partial differentiation IAC 2. Screening and differentiation in one assay This multiplex system using several tubes allows for QTPBD strips of 8 wells allows for the detection of the most relevant beer spoilage bacteria detection of the most relevant spoilage organisms and yeast in one assay. The tubes are pre-coated, for 12 samples in one assay (for blockcycler, qpcr therefore the handling is easier and yeasts will be devices using FAM, HEX, ROX). detected without an additional Lyticase lysis step (included). Well FAM HEX ROX 1 NTC NTC IAC 2 Enterobacteriacae P. anomala S. diastaticus 3 P. damnosus P. acidilactici/pentosaceus/parvulus/ inopinatus P. claussenii 4 Pectinatus spp. Megasphaera spp. L. rossiae 5 L. brevis/l.parabrevis/l.brevisimilis L. lindneri L. cas ei/l.paracasei 6 L. buchneri/l. parabuchneri L. collinoides/l. paracollinoides L. perolens/l. harbinensis 7 L. plantarum/l. paraplantarum L. coryniformis IAC 8 L. acetotolerans L. backii PTC Separate versions of this kit are available additionally for use with the following: For the Lightcycler LC 480, a kit containing white strips (QTPBD 0096 LC 480) and For the MyGo Pro a kit containing 0.1 ml tubes (QTPBD 0096 MG)

5 QTPYB 0096 In a mixture of groups and specific parameters two samples can be run on one strip, therefore 24 samples per kit can be analyzed for: Sample 1 FAM HEX ROX 1 Lactobacillus, Pediococcus Pediococcus Enterobactericae 1 Wild yeast 1 Bottom-fermented yeast Acetic acid bacteria 1 Wild yeast 2 Top-fermented yeast Internal amplification control 3. Detection of bacteria Several kits are available for the detection of specific bacteria. QTPHR 0050 Detection of two classes of hop resistance genes allows for additional risk assessment depending on the hopping grade of the beer hora/horc Detection shows presence of spoilage bacteria. hita/orf5 Increased risk if these resistance genes are present as wells Internal Control QTPA 0050 Detection of acidic acid bacteria Lactobacillus spp. and Pediococcus spp. Megasphaera and Pectinatus Hop resistance genes hora/horc Internal Control TPPMD 0050 Detection of strictly anaerobic Megasphaera and Pectinatus which are known as strong taste and off-flavor changing bacteria 4. Detection of yeasts Aside from screening and identification kits, several specific kits for single yeasts are available. A multiplex detection system for several wild yeasts is in development. GEN-IAL First-Beer Differentation Kit 5. Biofilm forming species The formation of Biofilm will protect spoilage microgransim from cleaning procedure. An early detection of Biofilm may prevent severe problems. The kit QTPBF detects the biofilm forming species Lactococcus lactis, Leuconostoc mesenteroides and Pichia anomala. GEN-IAL P1 Hyb Probe Screening LC 2.0 FRET

6 GEN-IAL products for beer analysis Product Description No. of tests/amount Art. No. DNA preparation GEN-IAL Simplex Easy DNA kit DNA preparation of beverage samples 100 preparations SE 0100 GEN-IAL QuickGEN* Sample preparation filtration DNA preparation of beverage samples, Filtration 100 preparations FSE 0100 GEN-IAL QuickGEN* without wash buffer GEN-IAL QuickGEN* Sample preparation centrifugation Bacteria & yeast DNA preparation with centrifugation without enrichment DNA preparation of beverage samples, preenrichment 100 preparations CSOW 0100 DNA preparation of beverage samples, centrifugation 100 preparations CSE 0100 Qualitative multiplex real-time PCR GEN-IAL First-Beer Differentiation PCR Kit GEN-IAL First-Beer Differentiation PCR Kit for LC 480 GEN-IAL QuickGEN* First-Beer Differentiation PCR Kit GEN-IAL QuickGEN* First-Beer Differentiation PCR Kit for LC 480 GEN-IAL QuickGEN* First-Beer Differentiation PCR Kit for MyGo Pro GEN-IAL First-Beer yeast and bacteria differentiation TaqMan GEN-IAL First-Beer yeast and bacteria differentiation TaqMan LC 480 GEN-IAL QuickGEN* First-Beer yeast and bacteria differentiation TaqMan MyGo Pro GEN-IAL P1 Hyb Probe Screening LC 2.0 FRET GEN-IAL P1 Screening TaqMan GEN-IAL QuickGEN* P1 Screening TaqMan GEN-IAL QuickGEN* P1 Screening TaqMan GEN-IAL P1 Screening Spartan DX-12 TaqMan (19 species) of relevant beer spoilers (19 species) of relevant beer spoilers for LC480 (19 species) of relevant beer spoilers (19 species) of relevant beer spoilers for LC480 (19 species) of relevant beer spoilers for MyGo Pro Multiplex detection and identification of beverage spoiling bacteria and yeasts Multiplex detection and identification of beverage spoiling bacteria and yeasts Multiplex detection and identification of beverage spoiling bacteria and yeasts for MyGo Pro DNA screening and differentiation of bacteria and yeasts for LC 2.0 DNA Screening and differentiation of beer spoiling bacteria and yeasts DNA screening and differentiation of beer spoiling bacteria and yeasts DNA screening and differentiation of beer spoiling bacteria and hop resistance genes DNA screening without differentiation of bacteria and yeasts 96 reactions TPBD reactions TPBD 0096 LC reactions QTPBD reactions QTPBD 0096 LC reactions QTPBD 0096 MG 96 reactions TPYB reactions TPYB 0096 LC reactions QTPYB 0096 MG 50 reactions PP1H 0050 LC reactions PP1T reactions QPP1T reactions QPP1HR reactions PP1T 0050 SP GEN-IAL P1OHScreening Spartan DX-12 TaqMan DNA screening without differentiation of bacteria 50 reactions PP1TOH 0050 SP GEN-IAL QuickGEN* First-Biofilm TaqMan GEN-IAL First Yeast Hyb Probe Screening LC 2.0 FRET Specific DNA detection of Lactococcus lactis, Leuconostoc mesenteroides and Pichia anomala 50 reactions QTPBF 0050 DNA screening and differentiation of yeasts 50 reactions PYHYB 0050 LC2.0 * QuickGEN kits deliver a rapid, easy one-step DNA lysis and amplification method. Pre-enriched samples as well as centrifuged or filtrated samples may be used with CSE 0100 or FSE 0100 and subsequent QuickGEN detection kits.

7 Product Description No. of tests/amount Art. No. Bacteria Qualitative real-time PCR GEN-IAL Pectinatus spp./megasphaera spp. TaqMan Resistance genes Specific DNA detection and differentiation of Pectinatus and Megasphaera Qualitative real-time PCR 50 reactions TPPMD GEN-IAL hop resistance genes hora and horc/hita and orf5 TaqMan GEN-IAL QuickGEN* hop resistance genes hora and horc/hita and orf5 TaqMan Specific DNA detection of hop resistance genes 50 reactions TPHR 0050 Specific DNA detection of hop resistance genes 50 reactions QTPHR 0050 Yeast Qualitative real-time PCR GEN-IAL Dekkera spp. Specific DNA detection of Dekkera spp. 50 reactions QTPPYD 0050 GEN-IAL Dekkera anomala TaqMan Specific DNA detection of Dekkera anomala 50 reactions TPYDA 0050 GEN-IAL Pichia anomala TaqMan Specific DNA detection of Pichia anomala 50 reactions TPYPA 0050 GEN-IAL Saccharomyces diastaticus TaqMan Specific DNA detection of Saccharomyces diastaticus 50 reactions TPYSD 0050 GEN-IAL Pichia membranaefaciens TaqMan Specific DNA detection of Pichia membranaefaciens 50 reactions TPYPM 0050 GEN-IAL Bottom fermented yeast TaqMan Specific DNA detection of bottom fermented yeast 50 reactions TPYUG 0050 GEN-IAL Top fermented yeast TaqMan Specific DNA detection of top fermented yeast 50 reactions TPYOG 0050 GEN-IAL accessories real-time PCR GEN-IAL Dekkera bruxellensis Standards DNA standards for Dekkera bruxellensis quantification cfu DBST 0100 Color Compensation Kit LC 480 Color compensation kit for multiplex assays 5 reactions PP1TCC 0005 Color Compensation Kit LC LightCycler Color compensation kit for multiplex assays 5 reactions CCFH 0005 Washing solution Washing solution for SEW ml WS 0100 * QuickGEN kits deliver a rapid, easy one-step DNA lysis and amplification method. Pre-enriched samples as well as centrifuged or filtrated samples may be used with CSE 0100 or FSE 0100 and subsequent QuickGEN detection kits.

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