RealLine Food Kit Turkey Detect

Transcription

1 Instruction for Use DETECTION KIT FOR DNA FROM ALL KIND OF TURKEY IN EXTREMELY PROCESSED AND CANNED FOOD AND IN OTHER PRODUCTS BY REAL-TIME-PCR This Kit is for Food Diagnostic only! CIB tests valid from: December 2016 Rev _EN Page 1 of 11

2 Explanation of symbols used in labelling LOT REF Batch code Catalogue number Expiry date Temperature limitation Σ Content of number of tests Consult instructions for use Manufacturer Keep out of sunlight BIORON Diagnostics GmbH Rheinhorststr Ludwigshafen (Germany) Phone Fax: Legals: Limited Product Warranty: This warranty limits our liability for the replacement of this product. No warranties of any kind, express or implied, including, without limitation, implied warranties of merchantability or fitness for a particular purpose, are provided. BIORON Diagnostics GmbH shall have no liability for any direct, indirect, consequential, or incidental damages arising out of the use, the results of use, or the inability to use this product. Trademarks: FAM and VIC are trademarks of Applera Corporation or its subsidiaries in the US and certain other countries. Lightcycler is a registered trademark of Roche, Germany. CIB50700 Page 2 of 11

3 TABLE OF CONTENT 1. INTENDED USE 4 2. PRINCIPLE OF THE METHOD 4 3. COMPONENTS OF THE KIT 4 4. STORAGE AND TRANSPORTATION OF THE KIT 4 5. ADDITIONAL DEVICES AND MATERIALS REQUIRED 5 6. GENERAL REMARKS AND PRECAUTIONS 5 7. PCR ANALYSIS AND PROTOCOL 6 8. INTERPRETATION OF RESULTS 9 9. TROUBLESHOOTING 11 ABBREVIATIONS USED IN THE IFU FOR THE KIT PC EPC ENC Positive Control Extraction Positive Control Extraction Negative Control This control is a species specific control for the kit and should be used in the dilution 1:100. This positive control should be done also during the extraction procedure, to ensure that extraction is functioning. This negative control should be done also during extraction procedure, to ensure that there is no contamination during the extraction protocol. CIB50700 Page 3 of 11

4 1. INTENDED USE The is for the use in laboratory with a real-time PCR system for the fast and easy identification of DNA from all kind of turkey in extremely processed and canned products, e.g. as in food and soaps. 2. PRINCIPLE OF THE METHOD The enables the identification of animal specific DNA by TaqMan technique. If products contain PCR capable DNA, animal specific PCR fragments are amplified and detected by real-time PCR. Specific PCR fragments are only detected if the samples contain the corresponding DNA. No PCR product synthesis is obtained with DNA isolates of other animal or plant species with food relevance. Detection limit of the : 0.1 % of Turkey DNA from total DNA 3. COMPONENTS OF THE KIT CIB50700 Colour Polymerase Mix 4 x 195 µl clear PP-Mix TURKEY 4 x 65 µl blue Control DNA TURKEY (2.5 ng / µl) 4 x 35 µl green 0.5 x TE 1.8 ml clear 4. STORAGE AND TRANSPORTATION OF THE KIT Store at ( ) C Transportation of the kit on dry ice or with iced blue packs Protect the kit from light, especially the tubes. Do not use kits with damaged inner packages and get in contact with BIORON Diagnostics GmbH. CIB50700 Page 4 of 11

5 5. ADDITIONAL DEVICES AND MATERIALS REQUIRED DNA Extraction Kit for food; we recommend the validated kits: RealLine Food DNA Extraction 1 (REF CIB50111) PureColumn DNA Extraction (REF BI88050) reactions tubes or plates and seals recommended for the usage in the real-time cycler pipettes and sterile filter tips 1.5 or 2.0 ml reaction tubes, sterile racks for tubes and a benchtop cooler Real-time PCR-cycler with FAM channel (~ 518 nm) 6. GENERAL REMARKS AND PRECAUTIONS The kits have to be used by experienced personnel only. Strictly follow the instruction manual for reliable results. Analyses with the RealLine Food Kit - Turkey Detect components are very sensitive and require a careful handling. Especially handling of positive controls should be done very carefully, with own pipettes and workspace. The use of filter tips is urgently recommended. Never use same tips for different samples. Wear disposable gloves and protective clothing according national regulations. The room, installation and equipment have to be kept absolutely clean. Due to sensitivity of the probe to light, please protect the reagents. In order to insure to have all liquid at the bottom of the reaction tube with Control DNA, PP-Mix, and Polymerase Mix centrifuge carefully. Do not use kit after expiry data. Do not pool reagents from different lots. To prevent thaw-freeze cycles aliquot the reagents and store at (-15-25) C. CIB50700 Page 5 of 11

6 7. PCR ANALYSIS AND PROTOCOL PCR analyses of unknown samples should be tested in doubles and with following controls: EPC: Positive Extraction Control of the DNA extraction: Expected is a positive PCR signal in FAM channel. ENC: Negative Extraction Control of the DNA extraction: Expected is a negative PCR results in FAM channel. PC: Positive PCR Control with Control DNA TURKEY Expected is a positive PCR signal in FAM. The Ct of the PC is the limit of detection LoD. 7.1 Settings of real-time PCR devices: General settings of Real-time PCR thermocycler: Cycling protocol: Pre-Incubation 15 min 95 C Amplification 15 sec 95 C 60 sec 60 C* Cycles: 40 Cooling 30 sec 40 C *Fluorescence detection Lid temperature: 100 C FAM: Channel for Turkey detection Settings of the Roche LightCycler 480 II: Detection Format: Dual Color Hydrolysis Probe / UPL Probe Cycling protocol: Pre-Incubation 15 min 95 C Amplification 15 sec 95 C Acquisition Mode: None 60 sec 60 C Acquisition Mode: Single Cycles: 40 Cooling 30 sec 40 C FAM: Channel for Turkey detection Analysis: Abs quant/ Fit points CIB50700 Page 6 of 11

7 7.2 Preparation of the PC Control: The PC is functioning as the Limit of detection (LoD) and is diluted 1:100: e.g.: 294 µl TE (0.5 x) + 3 µl Control DNA TURKEY 7.3 Preparation of the PCR premix: Calculate number of PCR reactions: Number of sample DNA isolates + ENC + EPC + PC Thaw all necessary tubes, store them on ice or in a benchtop cooler and protect from light. If you prepare less samples at once time, please aliquot the reagents and freeze at ( ) C. Label tubes or plate according the laboratory standard. Completion of the Master Mix: Component Pipetting Volume for Pipetting Volume for 1 reaction 10 reaction Polymerase Mix 7.5µl 75 µl PP-Mix TURKEY 2.5 µl 25 µl Pipetting of PCR reactions: Pipette calculated volumes of the Polymerase Mix and the PP-Mix TURKEY into the tubes and mix carefully Pipette 10 µl of this Master Mix to the tubes or plate for PCR Add 5 µl each of sample DNA isolates, and controls. Seal the plate or close tubes carefully to prevent contaminations. Optionally centrifuge the plate or tubes to prevent air bubbles. Place the PCR reaction mixes in the real-time cycler and run the program (see chapter 7.1). CIB50700 Page 7 of 11

8 Recommended reactions for the PCR assay: Sample 1 Isolate A Sample 1 Isolate B EPC ENC PC (LoD) Master Mix 10 µl 10 µl 10 µl 10 µl 10 µl Sample DNA 5 µl 5 µl EPC 5 µl ENC 5 µl PC (LoD) 5 µl Final volume 15 µl 15 µl 15 µl 15 µl 15 µl Abbreviations see p.2 CIB50700 Page 8 of 11

9 8. INTERPRETATION OF RESULTS 8.1. For Negative Control ENC: The program should not detect any signal in the FAM channel When Ct NC detects a signal in the FAM channel less than the Ct PC (LoD): this indicates the presence of a contamination (s. paragraph 8.4) 8.2. For Positive Controls PC and EPC: The program should detect an increase of the amplification signal of Turkey DNA in the FAM channel and determine the Ct of the PC. Note: The Ct PC is the value that is necessary for the evaluation of the samples as the Limit of detection LoD 8.3. For food samples: The sample is considered as positive, when the value of the Ct is lower or equal to the value of the LoD (Ct PC). The sample is considered as negative, when the value of the Ct is higher than Ct (PC) or not determined. Note: If samples with a Ct above the LoD show a clear sigmoidal curve, the food sample should be checked again. This samples are borderline and have to be treated as non-specific signals Contamination: In case of contamination all positive results of this individual PCR run are considered unreliable. Actions are required to identify and eliminate the source of contamination. Repeat the analysis of all samples of this run that were identified as positive. Samples that show negative results in this run should be considered as negative. CIB50700 Page 9 of 11

10 8.5. Overview of results: Result Sample 1 Isolate A Sample 1 Isolate B EPC ENC PC (LoD) sample is positive sample is negative Results are not unique different results * contamination of PCR Mix or during extraction wrong cycling conditions Master Mix is not functioning wrong Master Mix *see Troubleshooting chapter Example image of a real-time PCR run: Control DNA LoD Negative CIB50700 Page 10 of 11

11 9. TROUBLESHOOTING Problem Cause Corrective action ENC is positive contamination replace the reagents clean equipment and working space carefully No signals in the PC Polymerase Mix or PP-Mix is degraded replace kit avoid thaw-freeze cycels No signals in the PC device is not functioning check cycling conditions check functionality of device No signals in the PC no DNA added re-check the working steps Different results between doubles or triples samples result not unique pipette and mix carefully repeat extraction and PCR Ct Sample > Ct PC (LoD) and signalling curves are sigmoid sample is treated as borderline repeat PCR from extraction add trna** for limitation of false positive results to the extraction Signals with a Ct > Ct PC have to be treated very carefully und should be re-checked. Unspecific amplification can be caused by unknown animals, plant species and also from spices. ** Recommendation for the adding of trna for blocking of unspecific reactions with tubes and plastics. Add 250 ng trna to the extraction procedure of food material with low DNA content, like candies. CIB50700 Page 11 of 11