Salmonella enterica PCR Detection Kit (Meat Product) Product Insert
Contents
1. 0 C until DNA is extracted and ready for PCR amplification 1 Lysate Preparation amoaoop Vortex the enriched bacteria sample for 10 to 15 seconds or invert several times to mix Aliquot a maximum of 1 mL of enriched sample into a microcentrifuge tube Centrifuge at 14 000 x g 14 000 RPM for 3 minutes Remove the supernatant by pouring or pipetting Ensure that the pellet is not dislodged Resuspend the pellet in 400 uL of Digestion Buffer Add 10 uL of reconstituted Proteinase K to the resuspension and mix well by vortexing Incubate the lysate at 55 C for 45 minutes Mix the lysate occasionally by vortexing 2 Sample Binding to Column a After incubation add 40 uL of Binding Solution 10 uL of S enterica Isolation Control soC and 180 uL of 96 100 ethanol to the lysis mixture and mix by vortexing Note Ensure that the S enterica Isolation Control soC is added for subsequent control detection in the PCR protocol Centrifuge the column assembly for 3 minutes at 14 000 x g 14 000 RPM to bind the bacterial DNA Note If all the liquid does not pass through the column spin for an additional 2 minute at 14 000 x g 14 000 RPM If a small amount of liquid still remains on the top the column proceed to Step 3a with the addition of Wash Solution I 3 Column Wash a Apply 500 uL of Wash Solution I to the column and centrifuge for 2 minutes at 14 000 x g 14 000 RPM b Discard the flowthrough and reassembl2. Fax 905 227 1061 BIOTEK si CORPORATION Email techsupport norgenbiotek com gt 3430 Schmon Parkway K Thorold ON Canada L2V 4Y6 i Phone 866 667 4362 905 227 8848 Salmonella enterica PCR Detection Kit Meat Product Product Insert Product 32100 Salmonella enterica have emerged as significant foodborne pathogens that pose a serious public health problem The symptoms of salmonellosis may include diarrhea fever vomiting and abdominal cramps with elderly new born and immunocompromised individuals the most susceptible S enterica is a facultatively anaerobic Gram negative bacterium that could survive low temperatures and freezing The majority of the 1 3 billion annual cases of Salmonella caused human gastroenteritis result from ingestion of contaminated food products such as raw or undercooked meat seafood and eggs as well as raw or unpasteurized milk and dairy products Salmonella infections are also contracted following consumption of fresh fruits or vegetables that have been contaminated by infected fertilizer Principle of the Test and Product Description Norgen s Salmonella enterica PCR Detection Kit constituents a ready to use system for the isolation and the detection of S enterica using end point PCR The kit first allows for the enrichment and isolation of bacterial DNA from meat or other food samples using spin column chromatography based on Norgen s proprietary resin The DNA is isolated free from inhibitors a
3. PCR Master Mix 2x PCR Control Master Mix S enterica Isolation Control IsoC and S enterica Positive Control PosC are tested against predetermined specifications to ensure consistent product quality Product Use Limitations Norgen s S enterica PCR Detection Kit is designed for research purposes only It is not intended for human or diagnostic use Product Warranty and Satisfaction Guarantee NORGEN BIOTEK CORPORATION guarantees the performance of all products in the manner described in our product manual The customer must determine the suitability of the product for its particular use Safety Information Biosafety level 2 practices are recommended for works involving Salmonella enterica Ensure the appropriate containment equipment and facilities are used for activities involving cultures or potentially infectious clinical materials Ensure that a suitable lab coat disposable gloves and protective goggles are worn when working with chemicals For more information please consult the appropriate Material Safety Data Sheets MSDSs These are available as convenient PDF files online at www norgenbiotek com Guanidine salts form highly reactive compounds when combined with bleach thus care must be taken to properly dispose of any of these solutions If liquid containing these buffers is spilt clean with suitable laboratory detergent and water If the spilt liquid contains potentially infectious agents clean the affected area first wit
4. ded for proper interpretation of results e The recommended minimum number of DNA samples tested per PCR run is 6 e Using a lower volume from the sample than recommended may affect the sensitivity of S enterica Limit of Detection 1 Prepare the PCR for sample detection Set 1 using 2X S enterica Detection PCR Mastermix and control detection Set 2 using 2X PCR Conrtol Mastermix as shown in Table 1 below The recommended amount of sample DNA to be used is 2 5 uL However a volume between 1 and 5 uL of sample DNA may be used as template Ensure that one S enterica detection reaction and one control reaction is prepared for each DNA sample Adjust the final volume of the PCR reaction to 20 uL using the Nuclease Free Water provided Table 1 PCR Assay Preparation PCR Components Volume Per PCR Reaction Sample DNA 2 5 uL Nuclease Free Water 7 5 pL Total Volume 20 uL 2 For each PCR run prepare one positive control PCR as shown in Table 2 below Table 2 PCR Positive Control Preparation PCR Components Volume Per RT PCR Reaction Total Volume 20 uL 3 For each PCR run prepare one no template control PCR as shown in Table 3 below Table 3 PCR Negative Control Preparation PCR Components Volume Per PCR Reaction Nuclease Free Water 10 uL Total Volume 20 uL Therefore at a minimum each PCR run will contain 6 separate PCR reactions D Salmonella enterica PCR Assay Pro
5. e selection of stringent reaction conditions The primers were checked for possible homologies to all in GenBank published sequences by sequence comparison analysis The specific detectability of all relevant strains has thus been ensured by a database alignment and by PCR amplification with the following bacteria commonly found in contaminated food samples Ecoli Streptococcus agalatiae Streptococcus dysgalatiae Sterptococcus uberis Staphylococcus aureus Listeria monocytogenes G Linear Range e The linear range analytical measurement of Norgen s Salmonella enterica PCR Detection Kit was determined by analysing a dilution series of a S enterica quantification standard ranging from 1 x 10 cfu ul to 1 x 10 cfu ul e Each dilution has been tested in replicates n 4 using Norgen s Salmonella enterica PCR Detection Kit on 1X TAE 1 7 Agarose gel e The linear range of Norgen s Salmonella enterica PCR Detection Kit has been determined to cover concentrations from 1 x 10 cfu l to at least 1 x 10 cfu ul e Under the conditions of the Norgen s Salmonella enterica DNA Isolation procedure Norgen s Salmonella enterica PCR detection Kit covers a linear range from 1 000 cfu mL to at least 1 x 10 cfu mL in enriched samples Frequently Asked Questions 1 How many samples should be included per PCR run e Norgen s Salmonella enterica PCR Detection Kit is designed to test 24 samples For every 6 samples a n
6. e the column and the collection tube c Apply 500 uL of Wash Solution Il to the column and centrifuge again for 2 minutes at 14 000 x g 14 000 RPM Note Ensure the appropriate amount of ethanol has been added to Wash Solution Il d Discard the flowthrough and reassemble the column and the collection tube Centrifuge for 2 minutes at 14 000 x g 14 000 RPM to ensure the resin is completely dry e Discard the collection tube 4 DNA Elution a Transfer the spin column to a provided 1 7 mL Elution tube b Apply 75 uL of Elution Buffer to the column and centrifuge at 2 600 x g 6 000 RPM for 2 minutes c Spin for an additional 2 minutes at 14 000 x g 14 000 RPM to complete the DNA elution C Salmonella enterica PCR Assay Preparation Notes e Before use suitable amounts of all PCR components should be completely thawed at room temperature vortexed and centrifuged briefly e The amount of 2X S enterica Detection PCR Mastermix and 2X PCR Control Mastermix provided is enough for up to 32 PCR reactions 24 sample PCR 4 positive control PCR and 4 no template control PCR each e For each sample one PCR reaction using the 2X S enterica Detection PCR Mastermix and one PCR reaction using 2X PCR Control Mastermix should be set up in order to have a proper interpretation of the result e For every PCR run one reaction containing S enterica Positive Control S enterica PosC and one reaction as no template control must be inclu
7. gramming 1 Program the thermocylcer according to the program shown in Table 4 below 2 Run PCR Table 4 S enterica Assay Program PCR Cycle Step Temperature Duration Cycle 1 Step 1 95 C 3 min Step 1 94 C 15 sec Cycle 2 40x Step 2 60 C 30 sec Step 3 72 C 45 sec Cycle 3 Step 1 72 C 5 min Cycle 4 Step 1 4 C o E Salmonella enterica PCR Assay Results Interpretation 1 For the analysis of the PCR data the entire 15 20 uL PCR Reaction should be loaded on a 1X TAE 1 7 Agarose DNA gel along with 10 uL of Norgen s DNA Marker provided Prepare enough agarose gel for running one set of PCR of S enterica detection and one set of PCR for controls detection 2 The PCR products should be resolved on the 1X TAE 1 7 Agarose gel at 150V for 30 minutes Gel running time will be vary depending on an electrophoresis apparatus 3 Sample results are provided below M S enterica NC o OO 2000 1500 1000 750 500 300 _ S enterica Target 150 Figure 1 A representative 1X TAE 1 7 agarose gel showing the amplification of S enterica S enterica Target using the 2X S enterica Detection PCR Mastermix The size of the S enterica target amplicon corresponds to 336 bp as represented by the provided DNA Marker M NC Negative Control 2000 1500 1000 750 500 300 Isolation Control 150 lt lt PCR Control Figure 2 A representative 1X TAE 1 7 aga
8. h laboratory detergent and water and then with 1 v v sodium hypochlorite Protocol A Salmonella enterica Enrichment Important Notes Prior to Use e The following protocol is for preparing 1 L of selective media for S enterica enrichment which is enough for a total of 4 samples Each sample requires 225 mL of broth e Unused media should be stored at 4 C under sterile condition e All prepared enrichment broth should be used within 1 month after preparation 1 Salmonella enterica Enrichment Broth Preparation a Dissolve 54g of S enterica Enrichment Media in 1 L of distilled water b Ensure a final pH of 6 0 0 2 c Autoclave at 121 C for 15 minutes d Allow media to cool to room temperature before proceeding with Step 2 2 Salmonella enterica Enrichment from Food Samples a For meat or any solid food products weigh out 25 g of sample If the input is liquid such as milk aliquot 25 mL of sample b Add the sample to 225 mL S enterica Enrichment Broth as prepared above in A 1 c Incubate at 37 C for 24 hours d Proceed to S enterica Genomic DNA Isolation Section B B Salmonella enterica Genomic DNA Isolation Precaution All samples must be treated as potentially infectious material Important Notes Prior to Beginning Protocol e A variable speed centrifuge should be used for maximum kit performance If a variable speed centrifuge is not available a fixed speed centrifuge can be used however reduced yields ma
9. i G Amagliani G Brandi IJ Bruce and M Magnani 2009 Simultaneous Direct Detection of Salmonella spp Listeria monocytogenes and Escherichia coli 0157 in Milk Samples by Magnetic Extraction and Multiplex PCR Journal of Rapid Methods amp Automation in Microbiology 17 195 213 Related Products Product Milk Bacterial DNA Isolation Kit 21500 Bacterial Genomic DNA Isolation Kit 17900 Technical Assistance NORGEN s Technical Service Department is staffed by experienced scientists with extensive practical and theoretical expertise in sample and assay technologies and the use of NORGEN products If you have any questions or experience any difficulties regarding Norgen s Urine DNA Isolation Mini Kit Slurry Format or NORGEN products in general please do not hesitate to contact us NORGEN customers are a valuable source of information regarding advanced or specialized uses of our products This information is helpful to other scientists as well as to the researchers at NORGEN We therefore encourage you to contact us if you have any suggestions about product performance or new applications and techniques For technical assistance and more information please contact our Technical Support Team between the hours of 8 30 and 5 30 Eastern Standard Time at 905 227 8848 or Toll Free at 1 866 667 4362 or call one of the NORGEN local distributors www norgenbiotek com or through email at techsupport norgenbiotek com 3430 Schm
10. nd can then be used as the template in a PCR reaction for S enterica detection using the provided S enterica Master Mix The S enterica Master Mix contains reagents and enzymes for the specific amplification of a 336 bp region of the S enterica genome In addition Norgen s Salmonella enterica PCR Detection Kit contains a second Mastermix the PCR Control Master Mix which can be used to identify possible PCR inhibition and or inadequate isolation via a separate PCR reaction with the use of the provided PCR control PCRC or Isolation Control lsoC respectively This kit is designed to allow for the testing of 24 samples Kit Components Component Contents Salmonella a Enrichment 350g Lysis Solution 12 mL Binding Solution 4mL Wash Solution 15 mL Wash Solution II 5 mL Elution Buffer 8 mL Proteinase K 6 mg Mini Filter Spin Columns 25 Collection Tubes 25 Elution tubes 1 7 mL 25 Nuclease Free Water 1 25 mL Norgen s DNA Marker 0 1 mL Product Insert 1 IsoC Isolation Control PosC Positive Control The positive control is purified S enterica genomic DNA fragments The isolation control is a cloned PCR product Customer Supplied Reagents and Equipment e Disposable powder free gloves Benchtop microcentrifuge Micropipettors Sterile pipette tips with filters PCR tubes 96 100 ethanol 55 C incubator Storage Conditions and Product Stability All buffer
11. on Parkway Thorold ON Canada L2V 4Y6 Phone 905 227 8848 Fax 905 227 1061 Toll Free in North America 1 866 667 4362 2012 Norgen Biotek Corp PI32100 3
12. on template control and a Positive Control must be included It is preferable to pool and test 6 samples at a time If not the provided Positive Control is enough to run 3 samples at a time 2 How can interpret my results if neither the PCR control PCRC nor the Isolation Control IsoC amplifies e f neither the PCR control nor the Isolation Control amplifies the sample must be re tested If the positive control showed amplification then the problem occurred during the isolation where as if the Positive control did not amplify therefore the Problem has occurred during the setup of the PCR assay reaction 3 How should it be interpreted if only the PCR control PCRC showed amplification but neither the S enterica target nor the Isolation Control IsoC amplified for a sample e This indicates a poor isolation The isolation procedure must be repeated 4 How should it be interpreted if only the Isolation Control IsoC was amplified in a sample e The sample tested can be considered as S enterica negative 5 How should it be interpreted if only the S enterica target and the PCR control PCRC were amplified in a sample e The sample tested can be considered as S enterica positive 6 How should it be interpreted if only the S enterica target was amplified in a sample e The sample tested should be considered as S enterica positive At high S enterica cell input the S enterica amplicon will be predominant and thus the PCR con
13. rose gel showing the amplification of Isolation Control and PCR Conirol under different conditions using the 2X PCR Control Mastermix The size of the Isolation Control amplicon and PCR Control amplicon correspond to 499 bp and 150 bp respectively as represented by the provided DNA Marker M Lanes 1 to 5 showed detection of both Isolation Control and PCR Control suggesting that the DNA isolation as well as the PCR reaction was successful Lane 6 showed only the detection of PCR Control suggesting that while the PCR was successful the isolation failed to recover even the spiked in Isolation control NC Negative Control Table 5 Interpretation of PCR Assay Results Input Type Target Control Reaction Interpretation reaction Salmonella Salmonella Salmonella enterica enterica enterica Target Band IsoC Band PCRC Band 336 bp 499 bp 171 bp Positive xX xX xX Valid Control Negative Control x valig Sample X X X Positive Sample X X Negative Sample X Re test Sample Re test Sample X Negative Sample X X Positive Sample X X Positive Sample X Re test For results obtained that are not covered in Table 5 above please refer to the Troubleshooting Section F Salmonella enterica PCR Assay Specificity and Sensitivity The specificity of Norgen s Salmonella enterica PCR Detection Kit is first and foremost ensured by the selection of the S enterica specific primers as well as th
14. s should be kept tightly sealed and stored at room temperature 15 25 C Buffers can be stored for up to 1 year without showing any reduction in performance The Proteinase K should be stored at 20 C upon arrival and after reconstitution These reagents should remain stable for at least 1 year when stored at these conditions The S enterica 2x PCR Master Mix 2x PCR Control Master Mix S enterica Isolation Control lsoC and S enterica Positive Control PosC should be kept tightly sealed and stored at 20 C These can be stored for up to 1 year without showing any reduction in performance Repeated thawing and freezing gt 2 x of these reagents should be avoided as this may reduce the sensitivity If the reagents are to be used only intermittently they should be frozen in aliquots General Precautions The user should exercise the following precautions when using the kit e Use sterile pipette tips with filters e Store and extract positive material specimens controls and amplicons separately from all other reagents and add it to the reaction mix in a spatially separated facility e Thaw all components thoroughly at room temperature before starting an assay e When thawed mix the components and centrifuge briefly e Work quickly on ice Quality Control In accordance with Norgen s ISO 9001 and ISO 13485 certified Quality Management System each lot of Norgen s Salmonella enterica PCR Detection Kit including the S enterica 2x
15. trol PCRC as well as the Isolation Control IsoC may not amplify as they compete for PCR resources 7 How should it be interpreted if only the PCR control PCRC and the Isolation Control IsoC showed amplification in a sample e The sample tested can be considered negative 8 Can I freeze and thaw the provided enzymes for DNA isolation e Repeated freeze thaw of the reconstituted Proteinase K will reduce the activity of the enzymes and hence the isolation efficiency The result is lower DNA yield It is recommended to divide the reconstituted enzymes into smaller working aliquots prior to freezing 9 What If my incubation temperature during extraction varied from the specified 55 C for Proteinase K e At other temperatures the activity of the Proteinase K will be reduced This will result in a reduction in your DNA yields 10 What If my incubation time varied from the 45 minutes specified in the product manual e Less than 45 minutes will result in a lower DNA yields More than 45 minutes may not affect your DNA yields 11 What If I forgot to do a dry spin after my second wash e Your first DNA elution will be contaminated with the Wash Solution This may dilute the DNA yield in your first elution and it may interfere with the PCR detection as ethanol is known to be a PCR inhibitor 12 What If forgot to add Isolation Control IsoC during the Isolation e It is recommended that the isolation is repeated Reference E Omicciol
16. y be observed e Preheat an incubator or heating block to 55 C e Reconstitute the Proteinase K in 300 uL of molecular biology grade water aliquot into small fractions and store the unused portions at 20 C until needed e Prepare a working concentration of Wash Solution II by adding 15 mL of 96 100 ethanol to be provided by the user to the supplied bottle containing concentrated Wash Solution Il This will give a final volume of 20 mL The label on the bottle has a box that can be checked to indicate that ethanol has been added e S enterica Isolation Control soC A S enterica Isolation Control soC is supplied This allows the user to control the DNA isolation procedure For this assay add the S enterica Isolation Control IsoC to the lysate during the isolation procedure The S enterica Isolation Controk soC must not be added to the sample material directly Do not freeze and thaw the S enterica Isolation Control IsoC more than 2 times The S enterica Isolation Control IsoC must be kept on ice at all times during the isolation procedure e Bacterial Samples Immediate use of the enriched samples are recommended f the genomic DNA isolation is to be performed on a later date perform the protocol up to Step 1d Snap freeze the bacterial pellet with liquid nitrogen and store the pellet at 70 C until DNA Isolation e The PCR components of the Salmonella enterica PCR Detection Kit should remain at 2
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