DR. Food Kit User's Guide
Contents
1. 11 2 Extract genomic DNA from bacteria A platform for hybridization reaction Its surface is DR Food 12 pre spotted with a variety of specific probes for target Chip organisms Storage conditions Please store Part A at 20 C or below Part B at 2 8 C upon delivery DR Food Kit has demonstrated stability over a 9 month period when stored at recommended temperatures however we recommend to be discard the kit after expiration date Precaution gt Aliquot FM DNA Pol PC prior to use gt NOT leave DNA Pol at room temperature over 5 minutes appropriately trained molecular laboratory personnel shall operate this system All protocols in this manual shall be followed Gloves shall be worn at all time In case of contact with eyes rinse eyes immediately with plenty of water Do NOT touch the surface of the chip Before using E1 add 180 mL sterile water to obtain 200 mL 5 DR Food User s Guide 2 Mix 10 uL NBT BCIP with 490 uL Detection Buffer for each chip transfer the mixture to the hybridization chamber 3 Allow to react in the dark for 10 minutes at room temperature 4 Discard the detection liquid rinse the chip with 1 mL water twice and read the results of the pattern developed on the chip with DR AiM Reader 4 Results Pattern design principle Results are interpreted by the pattern formed on the chip Foodborne pathogen specific2. specific oligo probes with target DNA amplicons Wash Buffer Stringency wash to remove non binding amplicons after hybridization reaction Strep AP Using the specificity of biotin and streptavidin to detect the biotinylated molecules of amplicons The streptavidin conjugated alkaline phosphatase as enzyme to react with its substrate NBT BCIP for colorimetric signal on the chip Blocking Reagent Specifically formulated to reduce non specific nucleic acid binding on polymer chip surfaces also as a dilution buffer for Strep AP Detection Buffer A dilution buffer for NBT BCIP formulated to ensure Strep AP activity Staphylococcus Escherichia coli Listeria Salmonella spp Bacillus cereus aureus Shigella spp monocytogenes Yersinia als ae Positive Negative enterocolitica Vibrio spp Clostridium spp Control Control NBT BCIP Chromogenic phosphatase substrates Hydrolysis of 5 Bromo 4 chloro 3 indolyl phosphate indoly1 phosphate followed by oxidation produces a blue colored precipitate at the site of enzymatic activity NBT Nitro blue tetrazolium is the most commonly used electron transfer agent co precipitant for the BCIP reaction forming a dark blue precisely localized precipitate in the presence of Strep AP E1 Remove the PCR inhibitor for bacterial culture medium DR Food Kit User s Guide
3. oligonucleotide probes are designed and pre spotted on the chip to capture specific target amplicons After hybridization and colorimetric development perfectly matched probe target hybrid will form a blue purple precipitate on the chip as a result of enzymatic reaction Chip patterns are read and identified by DR AiM reader together with analysis software Refer to the DR AiM Reader user s guide Diagrams below show the patterns of the DR Food kit Note A successful test will show the relevant spots for Hybridization Positive Control and PCR Internal positive Control In the absence of the positive controls please check the operation process carefully DR Food Kit User s Guide V Hybridization Preparation for hybridization 1 DR Buffer Wash Buffer and chip to room temperature 2 Pre heat hybridization oven to 45 Hybridization Reaction 3 Pipette 25 uL amplicons into 500 uL pre warmed DR Hyb Buffer and mix thoroughly Denature mixture in boiling water for 10 minutes 5 Chill on ice for 3 minutes immediately and then spin down 6 Transfer 500uL of hybridization mixture onto the DR Food Chip incubate at 45 in the DR Mini Oven with maximum vibration for 1 hour VI Stringency Wash 1 Discard the hybridization liquid 2 Add 500uL Wash Buffer into the hybridization chamber and then discard the wash liquid Repeat this wash step three or four times 3 After st
4. 00 uL 1 Vial 2 DNA Pol 55 uL 1 vial 3 FM 1 3 mL 4 vials Part B Storage at 2 8 C No Descriptions Volume Quantity 1 DR Hyb Buffer 60 mL 1 bottle 2 Wash Buffer 200 mL 2 bottles 3 Strep AP 55 uL 1 vial 4 Blocking Reagent 60 mL 1 bottle 5 Detection Buffer 120 mL 1 bottle 6 NBT BCIP 1 2 mL 1 vial 7 1 10X concentrate 20 mL 1 bottle 8 E2 1 3 mL 4 vials 9 DR Food Chip 3 reactions 32 packs Description of products No Reagent Description 1 PC Positive control DNA template of PCR 2 DNA Pol PCR enzyme 5U pL DR Food Kit User s Guide Allocation of specific oligonucleotide probes Interpretation of results e Hybridization Positive Control e Staphylococcus aureus Salmonella spp Escherichia coli Shigella spp Listeria monocytogenes Bacillus cereus 9 Yersinia enterocolitica Vibrio spp e Clostridium spp PCR positive control Negative control DR Food Kit User s Guide FM PCR premix for amplifying specific target genes Staphylococcus aureus Escherichia coli Yersinia enterocolitica Clostridium perfringens Bacillus cereus Listeria monocytogenes Salmonella spp Shigella spp Vibrio spp and DNA internal control the reagent contains specific 5 biotinylated primer pairs dNTPs PCR buffer and MgCl DR Hyb Buffer Specially formulated to facilitate hybridization of
5. DR Food Kit 2 DR Chip Biotech No 31 Ke Jung Road Science Based Park Chu Nan Miao Li Taiwan 350 TEL 886 37 585 585 FAX 886 37 585 586 Email sales mail bio drchip com tw http www bio drchip com tw User s Guide DR Food Kit User s Guide DR Food Kit User s Guide 2 DR Chip Biotech No 31 Ke Jung Road Science Based Park Chu Nan Miao Li Taiwan 350 TEL 886 37 585 585 FAX 886 37 585 586 Email sales mail bio drchip com tw http www bio drchip com tw DME FP 0403 E DR Food Kit User s Guide DR Food Kit User s Guide DR Food Kit Notes User s Guide DR Food Kit Contents 1 Kit Contents Specifications Description of products Storage conditions Precaution 2 Introduction Product characteristics Limitations 3 Procedures and required materials Materials required but not provided Procedure Sampling amp preparation ll Enrichment lll DNA extraction IV DNA amplification V Hybridization VI Stringency wash VIl Colorimetric development 4 Results Pattern design principle Allocation of specific probes Interpretation of results User s Guide O A 00 004 HB HP HPW WRK DR Food Kit User s Guide DR Food Kit User s Guide 1 Kit Contents Notes Specifications Part A Storage at 20 C or below No Contents Volume Quantity 1 PC 3
6. ep 2 invert the chip chamber and repeatedly tap on paper towel to remove any residual liquid VII Colorimetric Development Blocking 1 For each chip mix 0 5 uL Strept AP with 500 uL Blocking Reagent transfer the mixture into the chamber allow to react for 30 minutes at 25 35 C 2 Discard the reacted blocking liquid 3 500uL Wash Buffer into the hybridization chamber and then discard the wash liquid Repeat this wash step three or four times 4 After step 3 invert the chip chamber and repeatedly tap on paper towel to remove any residual liquid Colorimetric Reaction 1 Rinse chip with 500 uL Detection Buffer and discard the liquid DR Food Kit User s Guide 2 Introduction Product Characteristics DR Food kit is a foodborne pathogen detection system for Staphylococcus aureus Escherichia coli Yersinia enterocolitica Bacillus cereus Clostridium spp Clo perfringens Clo butulinum Listeria monocytogenes Salmonella spp Shigella spp and Vibrio spp The system provides more d kit system applies four accurate test results than bacterial culture DR Foo molecular biology techniques DNA extraction PCR amplification DNA hybridization and colorimetric development using the following principle The genomic DNA of the pathogens is extracted from bacterial enrichment culture and the target gene is replicated using PCR amplification technology The amplicons are hybridized with specific p
7. nding upon the type of food Check national regulations for detailed methods of food sample preparation or use of the methods described in Food Sampling and Preparation of Sample Homogenate Bacterial Analytical Manual Online Chapter 1 http www cfsan fda gov ebam bam 1 html by FDA as reference Enrichment 1 Add 25 mL or 25 g food sample in 225 mL LB broth or other suitable medium incubate at 37 C 24 2 hours 2 Carry out ten fold dilution of this enrichment culture with sterile water before DNA extraction step DNA Extraction e Before using 1 1 add 180 mL sterile water to obtain 200 mL 1 Transfer 1 mL of diluent which was obtained from previous step into a microcentrifuge tube and centrifuge at 8 000 g 4 C for 5 minutes 2 Remove the supernatant resuspend pellet with 1 ml E1 and mix gently 3 Centrifuge at 8 000 g 4 C for 5 minutes DR Food Kit User s Guide 4 Discard the supernatant and resuspend the pellet with 50 uL E2 vortex until mixed well 5 Heat in boiling water for 10 minutes chill on ice for 2 minutes 6 Vortex the mixture and centrifuge at 8 000 g 4 C for 5 minutes 7 Take 50 uL supernatant containing the isolated DNA to a new 1 5 ml eppendorf tube ready for amplification The extracted DNA can be stored at 4 C and used within 2 days IV DNA Amplification 1 Preparation of PCR Master Mix Transfer the following reagents into a PCR tube and mix thoroughly For e
8. robes which are found on the chip After hybridization stringent washing removes non target DNA fragments and residual amplicons After subsequent colorimetric development patterns will form on the chip corresponded to the pathogen within the food sample Limitations DR Food kit is designed to detect nucleic acids of the foodborne pathogens Staphylococcus aureus Escherichia coli Yersinia enterocolitica Bacillus cereus Clostridium spp Clo perfringens and Clo butulinum Listeria monocytogenes Salmonella spp Shigella spp and Vibrio spp The detection limit is listed below for bacteria after enrichment Sensitivity Sensitivity Pathogen Pathogen cfu mL Staphylococcus aureus 107 107 Salmonella spp 107 104 Escherichia coli 10 10 Shigella spp 10 10 Yersinia enterocolitica 10 10 Vibrio spp 105 104 Bacillus cereus 10 10 Clostridium spp 10 10 Listeria monocytogenes 10 10 4 DR Food Kit User s Guide 3 Procedure and materials required Materials required but not provided Instruments gt Micropipettes lt Hotplate gt Microcentrifuge gt Thermal cycler Suggestion 100 Research Vortex mixer 20 C Freezer DR AiM Reader 2 DR Mini Oven Consumables tubes gt Tips 9 1 5 mL microcentrifuge tubes Procedure I Sampling amp Preparation Sampling methods and preparation vary depe
9. very batch of tests prepare additional tubes for PCR negative control and positive control Reagents Volume tube oftubes Total volume FM 395 1 9 uL DNA Pol 050 1 uL n number of sample including PCR negative control and positive control 2 Pipette 40 uL master mix into each PCR tube 3 Transfer 10 uL extracted DNA sample into each tube mix thoroughly for PCR negative control use 10 uL aseptic water and for PCR positive control use 10 uL PC positive control template reagent Final volume of each tube is 50 uL 4 Place all PCR tubes in a Thermal Cycler and follow the amplification program listed below 95 C 5min 95 C 30 60 C 30 30 cycles 72 C 30sec 72 C 5min 5 Proceed to hybridization reaction Ifnot used immediately please store amplicons at 20 C
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