STEC Detection Kits. Screening and Identification. For safer food BIOTECON Diagnostics simply builds up trust! Best Specificity.

Size: px
Start display at page:

Download "STEC Detection Kits. Screening and Identification. For safer food BIOTECON Diagnostics simply builds up trust! Best Specificity."

Transcription

1 foodproof STEC Detection Kits Screening and Identification For safer food BIOTECON Diagnostics simply builds up trust! Shigatoxin producing E. coli (STEC) like E. coli O157:H7, E. coli O157:H- or E. coli O104:H4 are known to cause different diseases like diarrhea, hemorrhagic colitis (HC) and the potentially fatal hemolytic uremic syndrome (HUS). STEC are most commonly transmitted through undercooked minced meat, unpasteurized or inadequately pasteurized milk, cold sandwiches, sprouts and vegetables. As most of these foods have relatively short shelf lives, the need for a rapid, accurate and sensitive detection method is a major food safety issue. The foodproof STEC Screening and Identification Kits are based on real-time PCR technology, which is well-established in the food industry as a highly sensitive and specific detection method. Fast: < 24 h to result with < 40 min of hands-on time Easy: Convenient, complete solution including DNA extraction and real-time PCR analysis Safe: Prevention of false-positive results and carry-over contamination Experienced: Manufacturer of by using Uracil-N-Glycosylase PCR-based rapid tests for the food industry since 1998 with an ISO Sensitive: Validated to detect accredited service lab 1 10 cfu/25 g sample, including enrichment Licensed: Fully licensed technology Best Specificity 100% Inclusivity STEC screening provides individual answers for stx1, stx2, and eae genes in one reaction 100% Exclusivity > 120* and > 50** strains tested, including closely related organisms and microorganisms of the same habitat Matrices > 100* tested food matrices, including meat, seafood, milk, fruits, vegetables and environmental samples Approvals* AOAC approved for All Kind of Foods * R , R ** R , R

2 Workflow Sample Enrichment DNA Extraction PCR Setup Real-time PCR Run Start approx. 30 min approx. 10 min min Analysis Results in < 2.5 h STEC Detection Kits R foodproof E. coli O157 Detection Kit (LC 1.x, 2.0) R foodproof E. coli O157 Detection Kit (5 Nuclease) R foodproof STEC Screening LyoKit (5 Nuclease) Detection of the virulence genes stx1, stx2 and eae R foodproof STEC Identification LyoKit (5 Nuclease) Identification of the 8 most important serotypes Safe and Specific The STEC Screening LyoKit detects stx1, stx2 and eae The stx2f variant is included, which will not be detected using the ISO TS method Specific DNA Extraction Kits KD-STEC-01 ENG V01/2014 S S S S foodproof ShortPrep II Kit - rapid purification foodproof Sample Preparation Kit I - high quality column purification foodproof StarPrep One Kit - bulk purification foodproof Magnetic Preparation Kit I - automated purification NEW LyoKits! Convenient, lyophilized, pre-filled reaction mix With internal amplification control and our proven UNG-System Easy storage Storage at -15 C to -25 C foodproof is a registered trademark of BIOTECON Diagnostics. The above mentioned real-time PCR instruments are registered trademarks of their respective holders. NEW! LyoKits storage at 2 C to 8 C 96 Reactions with final volume of 20 or 25 µl Instrument Compatibility LightCycler 5 Nuclease / TaqMan cycler (e.g. PikoReal 24, Mx3005P, CFX96, ABI 7500) BIOTECON Diagnostics GmbH Hermannswerder Potsdam Germany Phone: +49 (0) Fax: +49 (0) bcd@bc-diagnostics.com

3 FOR IN VITRO USE ONLY foodproof STEC Identification LyoKit 5 Nuclease Version 1, January 2015 PCR kit for the qualitative detection and simultaneous identification of Shiga toxin-producing E. coli (STEC) of the O-serotypes O26, O45, O103, O104, O111, O121, O145 and O157 using real-time PCR instruments. Order No. R / R Kit for 48 reactions (lyophilized) for a maximum of 46 samples Store the kit at 2 to 8 C 1

4 Table of contents 1. What this Product Does... 3 Number of Tests... 3 Storage and Stability... 3 Kit Contents... 3 Additional Equipment and Reagents Required How to Use this Product Before You Begin... 4 Precautions... 4 Sample Material... 4 DNA Extraction... 4 Positive Control... 4 Negative Control Procedure Data Interpretation Troubleshooting Additional Information on this Product... 8 How this Product Works... 8 Test Principle... 9 Prevention of Carry-Over Contamination... 9 Background Information... 9 References... 9 Quality Control Supplementary Information Ordering Information License Trademarks Contact and Support Change Index

5 1. 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 and negative control reactions can be analyzed per run. Storage and Stability Store the kit at 2 C to 8 C through the expiration date printed on the label. Once the kit is opened, store the kit components as described in the following Kit Contents table: Kit Contents Component Label Contents / Function / Storage foodproof STEC Identification LyoKit Microplate, prefilled with 48 reactions (lyophilized) Aluminum bag containing half a 8-tube strip mat R with white low profile tubes* R with clear regular profile tubes* 48 prefilled reactions (lyophilized). Ready-to-use PCR mix containing primer and probes specific for DNA of the eight serotypes and the Internal Control (IC) as well as Taq DNA Polymerase and Uracil-N-Glycosylase (UNG, heat labile) for prevention of carry-over contamination. For amplification and detection of specific sequences in the O- antigen gene clusters of O26, O45, O103, O104, O111, O121, O145, and O157. Store at 2 C to 8 C in the aluminum bag (sealed). Protect from light and moisture! Control Template Vial 2 (purple cap) 1 x 250 µl Contains a stabilized solution of DNA. For use as a PCR run positive control. Store at 2 to 8 C. H 2O PCR-grade Vial 3 (colorless cap) 1 x 1 ml Nuclease-free, PCR-grade H 2O. For use as a PCR run negative control. Store at 2 to 8 C. Cap strips Plastic bag containing 8-cap strips 12 x 8-cap strip For use in real-time PCR after addition of samples. *Tube profile and instrument compatibility chart is available online: Additional Equipment and Reagents Required Real-time PCR cycler suitable for detection of FAM-, HEX-, ROX- and Cy5-labeled probes and capable of performing a melting curve analysis. Without a melting curve analysis, the 8 serotypes can still be detected but not differentiated. In cases the strip tubes don t fit for the instrument the samples have to be transferred to appropriate PCR vessels after resuspension of the lyophilized PCR mix. Sample Preparation Kit foodproof StarPrep One Kit (Order No. S ) 1 or foodproof Magnetic Preparation Kit I (Order No. S L) 1 Nuclease-free, aerosol-resistant pipette tips Pipettes 1 Available from BIOTECON Diagnostics; see ordering Information for details Applicability Statement The foodproof STEC Identification LyoKit 5 Nuclease is intended for the rapid analysis of samples previously tested positive for Shiga toxin-producing E. coli DNA (e.g., with the foodproof STEC Identification LyoKit 5 Nuclease R ) isolated from enrichment cultures prepared by valid methods and inoculated with all relevant kinds of samples that are potentially contaminated with Shiga toxin-producing E. coli. The kit must not be used in diagnostic procedures. 3

6 The kit described in this Instruction Manual has been developed for real-time PCR instruments with a FAM, a HEX, a ROX and a Cy5 detection channel which are capable of performing a melting curve analysis. The performance of the kit was tested with the following real-time PCR instruments: LightCycler 480, LightCycler 96 (Roche Diagnostics), Mx3005P (Agilent Technologies), ABI 7500 (Applied Biosystems), iq5, CFX96 (Bio-Rad), and PikoReal 24 (Thermo Scientific). Note: A Color Compensation (Color Compensation Set 3; Order No. A ) is necessary and will be supplied by BIOTECON Diagnostics for users of the LC 480 Systems I and II. Please contact BIOTECON Diagnostics for further information. 2. How to Use this Product 2.1 Before You Begin Precautions Detection of DNA using the foodproof STEC Identification 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 nuclease-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 labware (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 STEC Identification lyophilized 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 sample enrichments, refer to the corresponding product package inserts of a suitable sample preparation kit (see Additional Equipment and Reagents Required ). DNA Extraction BIOTECON Diagnostics provides sample preparation kits suitable for all kind of food samples and PPS (see Additional Equipment and Reagents Required ). For additional testing of stx-positive samples, use the same DNA extract that was tested with the foodproof STEC Screening LyoKit. For more product information please refer to Positive Control Always run a positive control with the samples. To prepare a positive control, replace the template DNA with the provided control DNA [foodproof STEC Identification 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 H 2 O PCR-grade (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. 4

7 2.2 Procedure Program Setup The following procedure is optimized for a real-time PCR instrument with a FAM (amplification of the target serotypes), HEX (melting curve differentiation), ROX (melting curve differentiation) and Cy5 (amplification of the Internal Control and melting curve differentiation) detection channel. Program the PCR instrument before preparing the PCR samples. Use the following real-time PCR-protocol for the foodproof STEC Identification LyoKit. For details on how to program the experimental protocol, see the Instrument Operator s Manual of your real-time PCR-cycler: Pre-incubation Step 1: Step 2: Amplification Step 1: Step 2*: 1 cycle 37 C for 4 minutes 95 C for 5 minutes 50 cycles 95 C for 5 seconds 60 C for 60 seconds * Fluorescence detection in step 2 Melting Curve Step 1: Step 2: Step 3*: 1 cycle 95 C for 50 seconds 37 C for 50 seconds ramp up to 80 C * Fluorescence detection during C ramp with 1 2 measurements/ C Notes: For some real-time PCR instruments the type of the probe quencher as well as the usage of a passive reference dye has to be specified. The foodproof STEC Identification LyoKit contains probes with a non-fluorescent ( dark ) quencher and no passive reference dye. For users of the Agilent Mx3005P instrument: Choose Experiment Type SYBR Green (with Dissociation Curve) and add HEX, ROX, and CY5 channels for data collection in the setup section. Please contact BIOTECON Diagnostics, if you have questions how to program your cycler 5

8 Preparation of the PCR Mix Proceed as described below to prepare a 25 µl standard reaction. Always wear gloves when handling PCR strips or caps. Note: The lyophilizate is only stable in the provided aluminum bag with the silica gel pad. 1. Take the needed number of PCR tube strips out of the aluminum bag. Use a scissor or scalpel to cut the strips apart. Close the bag afterwards. 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. Decap the tube strips cautiously and discard the clear 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. Pipet 25 µl sample into each PCR-vessel and resuspend the pellet thoroughly by cautiously pipetting up and down: For the samples of interest, add 25 µl sample DNA (if you use less, then add to 25 µl with H 2 O PCR-grade). For the negative control, add 25 µl H 2 O PCR-grade (vial 3, colorless cap). For the positive control, add 25 µl foodproof STEC Identification Control Template (vial 2, purple cap). 5. Seal the vessels accurately and tightly with the colorless cap strips. Note: Alternatively resuspend the pellet after sealing by mixing thoroughly. To reduce the risk of crosscontamination, it is recommended to prepare only one PCR tube strip at a time. 6. Briefly spin the PCR tube strips in a suitable centrifuge. 7. Cycle the samples as described above. Note: For using the LightCycler 480 instrument a special adapter (Order No. Z ) 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 column 2 and 11. 6

9 2.3 Data Interpretation The kit is intended for further analysis of samples that were tested positive for stx1 and/or stx2 with the foodproof STEC Screening LyoKit to check for the presence of the serotypes O26, O45, O103, O104, O111, O121, O145, and O157. However, it detects E. coli of the target serotypes regardless of their pathogenic potential. Amplification The amplification of the O-serotype specific target genes is analyzed in the fluorescence channel suitable for FAM labeled probes detection. The specific amplification of the Internal Control is analyzed in the fluorescence channel suitable for Cy5. Compare the results from channel FAM and channel Cy5 for each sample, and interpret the results as described in the table below. Channel FAM Channel Cy5 Result Interpretation Positive Negative Positive or Negative Positive Negative Negative Invalid Positive for one or more of the following E. coli serotypes: O26, O45, O103, O104, O111, O121, O145, O157 Negative for STEC of the following E. coli serotypes: O26, O45, O103, O104, O111, O121, O145, O157 Note: The Control Template contains a mixture of all eight target sequences and therefore usually generates significantly higher fluorescent values than samples that are positive for only one or two of the target serotypes. This can affect positive/negative calls in automatic analysis of amplification curves by the respective instrument software. Always check results visually for plausibility. Melting curves Samples that are positive in the FAM detection channel can be further differentiated using a melting curve analysis in channels HEX, ROX, and Cy5. A prerequisite for the unambiguous discrimination of the serotypes in this multicolor experiment is a suitable calibration of the PCR instrument for channels FAM, HEX, ROX and Cy5. Please refer to the Operator s Manual of your real-time PCR cycler for further information. The following table lists the detectable serotypes in the respective channels and the typical melting peak temperature (Tm) range: Peak 1 Peak 2 Peak 3 Channel Serotype Tm Range Serotype Tm Range Serotype Tm Range HEX O C O C O C ROX O C O C O C Cy5 O C O C IC > 65 C Note: The melting peak temperature ranges given in the above table mainly reflect the variability between instruments and their respective analysis software. On a given instrument, the peaks in one channel have a distance of at least 3 C (e.g., on a LightCycler 480, the peak of O26 typically is at 45 C while the peak of O104 is at 50 C). The Control Template contains a mixture of all target sequences and can be used as a reference for the melting peak ranges in the particular PCR-run. However, food and sample preparation matrices can slightly shift melting peak temperatures. Melting peaks that do not fall into the specified Tm ranges can occur, but are not attributed to one of the target serotypes and can safely be ignored. For example, it has been observed that extracts of some food enrichments show a peak around 44 C in the Cy5 detection channel. The peak height of positive samples may vary according to the initial cell concentration. Note that the presence or absence of specific melting peaks should be checked manually for all positive samples as the peak finding algorithms of the respective PCR instrument software may not detect all relevant maxima of the melting curve. A guarantee for the identification via melting curves cannot be given. 7

10 3. Troubleshooting Observation Possible Reason Recommendation No signal increase is observed, even with positive controls. No signal increase in channel Cy5 is observed. Fluorescence intensity is too low. Strong decrease of fluorescence baseline Negative control samples are positive. Fluorescence intensity varies. Pellets are difficult to dissolve. Amplification positive and melting curve negative, or vice versa. Incorrect detection channel has been chosen. Pipetting errors. No data acquisition programmed. Inhibitory effects of the sample material (e.g., caused by insufficient purification). Inappropriate storage of kit components. Low initial amount of target DNA. Resuspension of lyophilized PCR mix not complete. Carry-over contamination. Insufficient centrifugation of the PCR strips. Resuspended PCR mix is still in the upper part of the vessel. Outer surface of the vessel or the seal is dirty (e.g., by direct skin contact). The lyophilized PCR mix started to rehydrate. Low initial amount of target DNA. Set channel settings to FAM. Check for correct reaction setup. Repeat the PCR run. Always run a positive control along with your samples. Check the cycle programs. Use the recommended DNA sample preparation kit to purify template DNA. Dilute samples or pipet a lower amount of sample DNA (e.g., 5 µl instead of 25 µl). Store the foodproof STEC Identification lyophilized PCR Mix at 2 C to 8 C, protected from light and moisture. Increase the amount of sample DNA. Depending on the chosen DNA isolation method, inhibitory effects may occur. Always resuspend lyophilized PCR mix thoroughly. 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. Always centrifuge PCR strips. Always wear gloves when handling the vessels and seal. Store the lyophilized PCR mix always in the aluminum bag with the silica gel pad. Open strip shortly before filling. Prolong the enrichment time and perform a new sample preparation. Increase the amount of sample DNA. Depending on the chosen DNA isolation method, inhibitory effects may occur. 4. Additional Information on this Product How this Product Works The foodproof STEC Identification 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, whereas the STEC DNA is detected in channel FAM. In case of a negative result due to inhibition of the amplification by the sample DNA of interest, the amplification of the IC is suppressed as well, whereas a negative result for the sample DNA of interest and amplification of the IC clearly indicates the absence of DNA of the target organisms in the sample. The foodproof STEC Identification LyoKit minimizes contamination risk and contains all reagents (except for template DNA) needed for the detection of DNA of the target serotypes. Primers and probes provide specific detection in food samples. The described performance of the kit is guaranteed for use on the real-time PCR instruments listed above only. 8

11 Test Principle 1. Using the kit's sequence-specific primers in a polymerase chain reaction (PCR), the PCR instrument and the supplied reagents amplify fragments of O-antigen gene specific sequences. 2. The PCR instrument detects these amplified fragments in real time through fluorescence generated by cleavage of the hybridized probes due to the 5 -nuclease activity of the Taq DNA polymerase. 3. During the ramp phase of the PCR (melting curve), additional probes hybridize to an internal sequence of the amplicons which 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-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 STEC 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 STEC Identification LyoKit, decontamination can be achieved with the provided reagents. Background Information Shiga toxin-producing Escherichia coli comprise strains of the bacterium Escherichia coli that, when infecting humans, have been linked with the severe complication hemolytic-uremic syndrome (HUS). They are known by a number of names, including enterohemorrhagic E. coli (EHEC), Shiga-like toxin-producing E. coli (STEC), hemolytic uremic syndrome associated enterohemorrhagic E. coli (HUSEC) and verocytotoxin- or verotoxinproducing E. coli (VTEC) [1]. Some STEC serotypes seem to be linked to severe complication like HUS more often than others: E. coli O157, accounting for 50 to 80 % of human STEC infections The Big Six (O26, O45, O103, O111, O121, O145), responsible for approximately 80 % non-o157 infections [2]. Under USDA regulation, E. coli serogroups O26, O103, O45, O111, O121 and O145 are prohibited from raw ground beef, its components, and tenderized steaks. E. coli O104, the cause of the 2011 Escherichia coli O104 outbreak. Identification of Shiga toxin-producing Escherichia coli with the foodproof STEC Identification LyoKit is in accordance with ISO/TS [3] and USDA-FSIS method MLG 5B [4]. References 1. Karch H, Tarr P, Bielaszewska M (2005). "Enterohaemorrhagic Escherichia coli in human medicine.". Int J Med Microbiol 295 (6-7): Gould LH, Mody RK, Ong KL, et al. Increased recognition of non-o157 Shiga toxin-producing Escherichia coli infections in the United States during : epidemiologic features and comparison with E. coli O157 infections. Foodborne Pathog Dis May;10(5): ISO/TS 13136:2012 Microbiology of food and animal feed Real-time polymerase chain reaction (PCR)-based method for the detection of food-borne pathogens Horizontal method for the detection of Shiga toxin-producing Escherichia coli (STEC) and the determination of O157, O111, O26, O103 and O145 serogroups. 4. FSIS Microbiology Laboratory Guidebook (MLG) 5B, Detection and Isolation of Non-O157 Shiga-Toxin Producing Escherichia coli (STEC) from Meat Products, Quality Control The foodproof STEC Identification LyoKit is function tested using the LightCycler 480 System (R ) and the Mx3005P (R ). 9

12 5. Supplementary Information 5.1 Ordering Information BIOTECON Diagnostics is offering a broad range of reagents and services. For a complete overview and for more information, please visit our website at License License Notice NOTICE TO PURCHASER: LIMITED LICENSE Use of this product is covered by one or more of the following US patents and corresponding patent claims outside the US: 5,804,375, 5,538,848, 5,723,591, 5,876,930, 6,030,787, 6,258,569. The purchase of this product includes a limited, non-transferable immunity from suit under the foregoing patent claims for using only this amount of product solely in Food Testing Applications and Genetically Modified Organism (GMO) Testing Applications, including reporting results of purchaser s activities for a fee or other commercial consideration, and also for the purchaser's own internal research. No right under any other patent claim is conveyed expressly, by implication, or by estoppel. Further information on purchasing licenses may be obtained from the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA. The purchase price of this product includes limited, nontransferable rights under U.S. 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 outlicensing@lifetech.com. 5.3 Trademarks foodproof is a trademark of BIOTECON Diagnostics GmbH. Other brand or product names are trademarks of their respective holders. 5.4 Contact and Support If you have questions about this or any other product of BIOTECON Diagnostics, please contact our Technical Support staff (for details see 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. 6. Change Index Version 1, January 2015: First version of the package insert. R (1) BIOTECON Diagnostics GmbH Hermannswerder Potsdam Germany Phone +49 (0) Fax +49 (0) bcd@bc-diagnostics.com 10

13 foodproof L. monocytogenes Detection Kits Hybridization and Hydrolysis Probes For safer food BIOTECON Diagnostics simply builds up trust! Listeria monocytogenes is known to be responsible for listeriosis, a rare but often lethal food-borne infection with a lethality rate of 25%. Usually Listeria is transmitted through contaminated food, such as raw milk, cheeses, raw and cooked poultry, raw meats, ice cream, raw or smoked fish and raw vegetables. Listeria is very resistant and able to grow in temperatures ranging from 4 C to 37 C. The foodproof Listeria monocytogenes Detection Kit is based on the real-time PCR technology which is well-established in the food industry as a highly sensitive and specific detection method. Fast: h to result with < 40 min of hands-on time Safe: Prevention of false-negative results by internal control and prevention of carry-over contamination using Uracil-N- Glycosylase Easy: Convenient, complete solution including DNA extraction and real-time PCR analysis Experienced: Manufacturer of PCR-based rapid tests for the food industry since 1998 with an ISO accredited service lab Best Specificity 100% Inclusivity > 100 L. monocytogenes strains tested 60 strains tested including closely related organisms and microorganisms of the same habitat Matrices All relevant food matrices tested, including milk, cheeses, poultry, ice cream, spices, minced meat and environmental samples Approvals Sensitive: Validated to detect 1 10 cfu/25g sample, including enrichment Licensed: Fully licensed technology AOAC approved for a variety of foods

14 Workflow Enrichment Start DNA Extraction approx. 30 min PCR Setup approx. 10 min Real-time PCR Run min Analysis Results in < 2,5 h Listeria monocytogenes Detection Kits R foodproof L. monocytogenes Detection Kit (LC 1.x, 2.0) R foodproof L. monocytogenes Detection Kit (5 Nuclease) Specific DNA Extraction Kits S foodproof ShortPrep II Kit - rapid purification S foodproof Sample Preparation Kit II - high quality column purification S foodproof StarPrep Two Kit - bulk purification S L foodproof Magnetic Preparation Kit II - automated purification DNA Extraction foodproof ShortPrep II Kit enables the extraction of Listeria monocytogenes DNA from food matrix enrichments Instrument Compatibility LightCycler 480 II 5 Nuclease / TaqMan cycler (e.g. PikoReal 24, Mx3005P, CFX96, ABI 7500) BIOTECON Diagnostics GmbH KD-LIM-01-EN V01/2014 Storage at -15 C to -25 C 96 Reactions with a final volume of 20 or 25 µl foodproof is a registered trademark of BIOTECON Diagnostics. The above mentioned real-time PCR instruments are registered trademarks of their respective holders. Hermannswerder Potsdam Germany Phone: +49 (0) Fax: +49 (0) bcd@bc-diagnostics.com

15 For food testing purposes FOR IN VITRO USE ONLY foodproof Alicyclobacillus Detection Kit, 5 Nuclease Identification of Alicyclobacillus acidoterrestris Version 1, May 2011 PCR kit for the qualitative detection of Alicyclobacillus DNA with simultaneous identification of Alicyclobacillus acidoterrestris DNA using real-time PCR instruments. Order No. R Kit for 96 reactions for a maximum of 94 samples Store the kit at -15 to -25 C 1

16 Table of contents 1. What this Product Does... 3 Number of Tests... 3 Storage and Stability... 3 Kit Contents... 3 Additional Equipment and Reagents Required How to Use this Product Before You Begin... 4 Precautions... 4 Sample Material... 4 DNA Extraction... 4 Positive Control... 5 Negative Control Procedure Data Interpretation Troubleshooting Additional Information on this Product... 7 How this Product Works... 7 Test Principle... 7 Prevention of Carry-Over Contamination... 7 Background Information... 7 Product Specifications... 7 References... 8 Quality Control Supplementary Information Ordering Information License Trademarks Contact and Support Change Index

17 1. What this Product Does Number of Tests The kit is designed for 96 reactions with a final reaction volume of 25 µl each. Up to 94 samples (single sample preparation) plus positive and negative control reactions can be analyzed per run. Storage and Stability Store the kit at 15 C to 25 C through the expiration date printed on the label. Once the kit is opened, store the kit components as described in the following Kit Contents table: Kit Contents Vial Label Contents / Function / Storage 1 yellow cap foodproof Alicyclobacillus Master Mix 2 red cap foodproof Alicyclobacillus Enzyme Solution 3 white cap foodproof Alicyclobacillus Internal Control 4 purple cap foodproof Alicyclobacillus Control Template 3 x 600 µl Ready-to-use primer and hydrolysis probe mix specific for Alicyclobacillus DNA and the Internal Control (IC). For amplification and detection of Alicyclobacillus-specific sequences. Store at -15 to - 25 C. Avoid repeated freezing and thawing! Protect from light! 3 x 32 µl Contains Taq DNA Polymerase and Uracil-DNA N-Glycosylase (UNG, heat labile) for prevention of carry-over contamination. Store at -15 to -25 C. 3 x 32 µl Contains a stabilized solution of plasmid DNA and a yellow dye for better visualization. For use as an internal amplification control. Store at -15 to -25 C. After first thawing store at +2 C to +8 C for up to one month. 1 x 50 µl Contains a stabilized solution of plasmid DNA. For use as a PCR run positive control. Store at -15 to -25 C. After first thawing store at +2 C to +8 C for up to one month. 5 colorless cap H2O, PCR-grade 1 x 1 ml Nuclease-free, PCR-grade H2O. For use as a PCR run negative control. Store at -15 to -25 C. 3

18 Additional Equipment and Reagents Required real-time PCR cycler suitable for detection of FAM-, VIC/HEX-, and ROX/Texas Red-labeled probes real-time PCR compatible tubes, strips or plates with optical cap or foil applicable for the PCR cycler in use foodproof ShortPrep II Kit 1 (Order No. S ) or foodproof Sample Preparation Kit II 1 (Order No. S ) Nuclease-free, aerosol-resistant pipette tips Pipettes Sterile reaction tubes for preparing PCR mixes and dilutions 1 Available from BIOTECON Diagnostics; see Ordering Information for details Applicability Statement The foodproof Alicyclobacillus Detection Kit 5 Nuclease is intended for the rapid detection of Alicyclobacillus DNA including the simultaneous identification of Alicyclobacillus acidoterrestris DNA isolated from enrichment cultures prepared by valid methods and inoculated with all kinds of foods that are potentially contaminated with Alicyclobacillus spp. The kit must not be used in diagnostic procedures. The performance of the kit described in this Instruction Manual is guaranteed only when it is used with real-time PCR instruments suitable for detection of FAM-, VIC/HEX-, and ROX/Texas Red-labeled probes, e.g.: LightCycler 480 (Roche Diagnostics), ABI 7500 (Applied Biosystems), icycler iq5 (BioRad), Mx3000/Mx3005 (Stratagene) or Rotorgene 6000 (Corbett Life Science). Note: A color compensation (Color Compensation Set 3; Order No. A ) is necessary and will be supplied by BIOTECON Diagnostics for users of the LC 480 Systems I and II. Please contact BIOTECON Diagnostics for further information. 2. How to Use this Product 2.1 Before You Begin Precautions Detection of Alicyclobacillus DNA using the foodproof Alicyclobacillus Detection Kit requires DNA amplification by PCR. The detection kit provides all the reagents required for the PCR. However, in order to achieve reliable results, the entire assay procedure must be performed under nuclease-free conditions. Follow the instructions below to avoid nuclease-, carry-over-, or cross-contamination: Prepare appropriate aliquots of the kit solutions and keep them separate from other reagents in the laboratory. Use nuclease-free labware (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 Alicyclobacillus Master Mix (vial 1, yellow cap) away from light. Sample Material Use any sample material suitable for PCR in terms of purity, concentration, and absence of inhibitors. For preparation of genomic DNA from raw material or from food enrichments, refer to the corresponding product package inserts of a suitable sample preparation kit (see Additional Equipment and Reagents Required ). DNA Extraction BIOTECON Diagnostics provides sample preparation kits suitable for all kind of foods and raw materials (see Additional Equipment and Reagents Required ). For more product information please refer to 4

19 Positive Control Always run a positive control with the samples. To prepare a positive control, replace the template DNA with the provided control DNA [foodproof Alicyclobacillus Control Template (vial 4, 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 H 2O, PCR-grade (vial 5, 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. 2.2 Procedure Program Setup Program the PCR instrument before preparing the reaction mixes. The amplification is carried out according to the following temperature-time-program (for details on how to program the experimental protocol, see the operation manual of your real-time PCR cycler): Pre-incubation Step 1: Step 2: Amplification Step 1: Step 2*: 1 cycle 37 C for 4 minutes 95 C for 5 minutes 50 cycles 95 C for 5 seconds 60 C for 60 seconds, For some real-time PCR instruments the type of the probe quencher as well as the usage of a passive reference dye has to be specified. The foodproof Alicyclobacillus Detection Kit contains probes with a non fluorescent ( dark ) quencher and no passive reference dye. NOTE for users of the Agilent Mx3005P instrument: Click Instrument Filter Set Gain Settings to open the Filter Set Gain Settings dialog box in which the gain settings may be viewed and modified. For FAM the Filter Set Gain Setting has to be modified to x1. * Fluorescence detection in step 2 Preparation of the PCR Mix Proceed as described below to prepare a 25 µl standard reaction. Always wear gloves when handling the PCR vessels. 1. Thaw the solutions and, for maximal recovery of contents, briefly spin vials in a microcentrifuge before opening. Mix carefully but thoroughly by pipetting up and down. 2. In a reaction tube ( ml, depending on the number of reactions), prepare the PCR mix by adding the following components in the order mentioned below, then mix gently but thoroughly by pipetting up and down. The volumes indicated below are based on a single 25 µl standard reaction. Prepare the PCR mix by multiplying the amount in the Volume column by the number of reactions (sample and control reactions) to be cycled plus one or two additional reactions to cover pipetting losses. Component Volume foodproof Alicyclobacillus Master Mix, (vial 1, yellow cap) 18.0 µl foodproof Alicyclobacillus Enzyme Solution, (vial 2, red cap) 1.0 µl foodproof Alicyclobacillus Internal Control, (vial 3, white cap) 1.0 µl Total volume 20.0 µl 3. Pipet 20 µl PCR mix into each PCR vessel. For the samples of interest, add up to 5 µl sample DNA (if less than 5 µl add H 2O to 5 µl). For the negative control, add 5 µl H 2O, PCR-grade (vial 5, colorless cap). For the positive control, add 5 µl foodproof Alicyclobacillus Control Template (vial 4, purple cap). 4. Seal the PCR vessels accurately with optical caps or sealing foil. 5. Briefly spin the PCR vessels in a suitable centrifuge. 6. Cycle the samples as described above. 5

20 2.3 Data Interpretation The amplification of DNA of Alicyclobacillus acidoterrestris is analyzed in the fluorescence channel suitable for FAM labeled probes detection. The amplification of DNA of all Alicyclobacillus species (including A. acidoterrestris) is analyzed in the fluorescence channel suitable for the detection of VIC/HEX labeled probes. The specific amplification of the Internal Control is analyzed in the fluorescence channel suitable for ROX/Texas Red. Compare the results from channel FAM (Alicyclobacillus acidoterrestris), channel VIC/HEX (Alicyclobacillus spp.) and channel ROX/Texas Red (Internal Control) for each sample, and interpret the results as described in the table below. Channel FAM Channel VIC/HEX Channel ROX/Texas Red Result Interpretation Positive Positive Positive or Negative Positive for Alicyclobacillus acidoterrestris Negative Positive Positive or Negative Positive for Alicyclobacillus spp. other than Alicyclobacillus acidoterrestris Negative Negative Positive Negative for Alicyclobacillus spp. (including Alicyclobacillus acidoterrestris) Negative Negative Negative Invalid Note: A prerequisite for the unambiguous discrimination of Alicyclobacillus and A. acidoterrestris DNA and Internal Control DNA in this multi-color experiment is a suitable calibration of the PCR instrument for channels FAM, VIC/HEX and ROX/Texas Red. Please refer to the operation manual of your real-time PCR cycler for further information. 3. Troubleshooting Observation Possible Reason Recommendation No signal increase is observed, even with positive controls. No signal increase in channel ROX/Texas Red is observed. Incorrect detection channel has been chosen. Pipetting errors or omitted reagents. No data acquisition programmed. Inhibitory effects of the sample material (e.g., caused by insufficient purification). Set Channel settings to FAM, VIC/HEX or ROX/Texas Red. Check for correct pipetting scheme and reaction setup. Repeat the PCR run. Always run a positive control along with your samples. Check the cycle programs. Use the recommended DNA sample preparation kit to purify template DNA. Dilute samples or pipet a lower amount of sample DNA (e.g., 2.5 µl instead of 5 µl). Fluorescence intensity is too low. Negative control samples are positive. Fluorescence intensity varies. Inappropriate storage of kit components. foodproof Alicyclobacillus Master Mix (vial 1, yellow cap) is not homogeneously mixed. Low initial amount of target DNA. Carry-over contamination. Insufficient centrifugation of the PCR vessels. Prepared PCR mix is still in the upper part of the vessel. Outer surface of the vessel or the seal is dirty (e.g., by direct skin contact). Store the foodproof Alicyclobacillus Master Mix (vial 1, yellow cap) at -15 C to -25 C, protected from light. Avoid repeated freezing and thawing. Mix the foodproof Alicyclobacillus Master Mix (vial 1, yellow cap) and the entire PCR-mix thoroughly before pipetting. Increase the amount of sample DNA. Depending on the chosen DNA isolation method, inhibitory effects may occur. 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. Always centrifuge reaction vessels. Always wear gloves when handling the vessels and seal. 6

21 4. Additional Information on this Product How this Product Works The foodproof Alicyclobacillus Detection Kit provides primers and hydrolysis probes (for sequence-specific detection), convenient premixed 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 supplied with the kit (vial 3, white cap). The IC has to be added to each reaction. A hydrolysis probe was designed to bind specifically the IC, allowing detection in the ROX/Texas Red channel, whereas the Alicyclobacillus DNA is detected in channels FAM (A. acidoterrestris) and VIC/HEX (all Alicyclobacillus spp.). In case of a negative result due to inhibition of the amplification by the sample DNA of interest, the amplification of the IC is suppressed as well, whereas a negative result for the sample DNA of interest and amplification of the IC clearly indicates the absence of Alicyclobacillus DNA in the sample. The foodproof Alicyclobacillus Detection Kit minimizes contamination risk and contains all reagents (except for template DNA) needed for the detection of Alicyclobacillus DNA. Primers and probes provide specific detection of Alicyclobacillus DNA in food 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 polymerase chain reaction (PCR), the PCR instrument and the supplied reagents amplify fragments of Alicyclobacillus spp. specific sequences. 2. The PCR instrument detects these amplified fragments in real time through fluorescence generated by cleavage of the hybridized probe due to the 5 -nuclease activity of the Taq DNA polymerase. The probe is labeled at the 5 -end with a reporter fluorophore and at the 3 -end with a quencher. 3. During the annealing/elongation phase of each PCR cycle, the probe hybridizes to an internal sequence of the amplicon and is cleaved by the 5 nuclease activity of the Taq DNA polymerase. 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 PCR s. 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 Alicyclobacillus 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 Alicyclobacillus Detection Kit, decontamination can be achieved with the provided reagents. Background Information Alicyclobacillus spp. are Gram-positive, rod-shaped, thermophilic and acidophilic, spore-forming bacteria. Depending on the different species, growth temperatures range from C, with optimums from C. Alicyclobacillus spp. can also grow over a wide ph range, generally reported between ph 2.5 and 6.0. The spores are present almost everywhere in the environment and can be brought into the company on contaminated fruit. It is one of the microorganisms of concern in the fruit juice industry. The thermophilic and acidophilic characteristics of Alicyclobacillus spp. allow resistance to current pasteurization processes, and the ability to produce off-flavors in juice poses potential economic losses for the juice industry. Most studies concerning Alicyclobacillus spp. related spoilage is focused on A. acidoterrestris. Guaiacol and halophenols were identified as the offensive smelling agents in many Alicyclobacillus spp. related spoilage (1). Since conventional microbiological methods for the detection and identification of Alicyclobacillus are very time-consuming, PCR has been introduced to the food industry as a highly sensitive and specific detection method (2). Product Specifications Specificity Inclusivity of the foodproof Alicyclobacillus Detection Kit has been tested with 38 strains of the genus Alicyclobacillus, including the following species: A. acidiphilus, A. acidocaldarius, A. acidoterrestris (7 strains), A. contaminans, A. cycloheptanicus, A. disulfidooxidans, A. fastidiosus, A. herbarius, A. hesperidum, A. kakegawensis, A. macrosporangiidus, A. pomorum, A. sacchari, and A. sendaiensis. All strains of A. acidoterrestris were detected in channels FAM and VIC/HEX, and all strains of other species than A. acidoterrestris were detected in channel VIC/HEX only. Exclusivity of the foodproof Alicyclobacillus Detection Kit has been tested with 40 strains of closely related genera (mainly of the order Bacillales). None of them was detected in channel FAM or channel VIC/HEX. Sensitivity The foodproof Alicyclobacillus Detection Kit detects down to cfu/ml in enrichment cultures (depending on the sample preparation kit used: foodproof ShortPrep II or foodproof Sample Preparation Kit II, respectively). 7

22 References 1. Chang S, Kang D (2004) Alicyclobacillus spp. in the fruit juice industry: History, characteristics, and current isolation/detection procedures. Critical Reviews in Microbiology 30, Scheu PM et al (1998) Detection of pathogenic and spoilage micro-organisms in food with the polymerase chain reaction. Food Microbiology 15, Quality Control The foodproof Alicyclobacillus Detection Kit is function tested using the LightCycler 480 System. 5. Supplementary Information 5.1 Ordering Information BIOTECON Diagnostics is offering a broad range of reagents and services. For a complete overview and for more information, please visit our website at License License Notice Use of this product is covered by one or more of the following US patents and corresponding patent claims outside the US:, 5,804,375, 5,538,848, 5,723,591, 5,876,930, 6,030,787, 6,258,569. The purchase of this product includes a limited, nontransferable immunity from suit under the foregoing patent claims for using only this amount of product solely in Food Testing Applications and Genetically Modified Organism (GMO) Testing Applications, including reporting results of purchaser s activities for a fee or other commercial consideration, and also for the purchaser's own internal research. No right under any other patent claim is conveyed expressly, by implication, or by estoppel. Further information on purchasing licenses may be obtained from the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA. The purchase price of this product includes limited, nontransferable rights under U.S. 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 outlicensing@lifetech.com. 5.3 Trademarks foodproof is a trademark of BIOTECON Diagnostics GmbH. Other brand or product names are trademarks of their respective holders. 5.4 Contact and Support If you have questions about this or any other product of BIOTECON Diagnostics, please contact our Technical Support staff (for details see 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. 6. Change Index Version 1, First version of the package insert. R (3) BIOTECON Diagnostics GmbH Hermannswerder Potsdam Germany Phone +49 (0) Fax +49 (0) bcd@bc-diagnostics.com 8

23 foodproof Norovirus Detection Kit Hydrolysis Probes For safer food BIOTECON Diagnostics simply builds up trust! Norovirus is an ubiquitous pathogen that causes gastroenteritis worldwide. The pathogen is a RNA virus belonging to the virus family Caliciviridae. As a highly contagious virus, there are often major outbreaks, particularly in facilities such as child daycares, schools or hospitals. Symptoms may include diarrhea, nausea, stomach cramps and vomiting. Severe cases have been reported caused by contaminated fruits, berries, raw vegetable, raw meat and shellfish. The foodproof Norovirus Detection Kit (GI, GII, GIV) is designed for an one-step real-time reverse transcriptase PCR for the simultaneous, qualitative detection, and differentiation of norovirus of the genogroup I, II and IV. Based on primers and probes of the ISO/TS 15216, and the 64 German Food and Feed Code (LFGB), the kit contains an additional phage process control. Specificity 100% specificity, 11 different virus species have been tested, comprising several virus families with RNA and DNA genomes. Sensitivity Limit of detection (LOD) was set at 10 virus copies per reaction for GI- and GII-RNA. Fast: < 3 h to result, easy-to-use mastermix. Safe: 3 controls included - positive, negative and process control. ISO/TS compliance: The Process Control (solution of MS2 phage) has to be added to the start of the sample processing. Easy: Add 10 µl RNA sample in one step to the mastermix. Experienced: Manufacturer of PCR based rapid tests for the food industry since 1998 with an ISO accredited service lab. Licensed: Fully licensed technology. Matrices Multiple food matrices tested, including berries, fruits, shellfish (incl. Pacific oysters, mussels), minced meat. In addition, stool samples tested positive.

24 Workflow Analysis Results in 3 h Start Virus concentration RNA Extraction approx. 45 min PCR Setup approx. 10 min Real-time PCR Run 140 min Norovirus real-time PCR Kit R foodproof Norovirus Detection Kit (GI, GII, GIV) Additional real-time PCR kits (of interest for seafood and berries) RDK foodproof Vibrio Detection LyoKit R foodproof Salmonella Detection LyoKit RNA Extraction foodproof Virus Sample Preparation Kit Optimized for and validated with the Norovirus Detection Kit Instrument Compatibility 5 Nuclease / TaqMan cycler (e.g. PikoReal 24, AriaMx, LightCycler 480, LC 96, ABI 7500) KD-NV-01-EN V01/2015 foodproof is a registered trademark of BIOTECON Diagnostics. The above mentioned real-time PCR instruments are registered trademarks of their respective holders. Storage at -15 C to -25 C 64 Reactions with a final volume of 25 µl BIOTECON Diagnostics GmbH Hermannswerder Potsdam Germany Phone: +49 (0) Fax: +49 (0) bcd@bc-diagnostics.com