E. coli O157:H7 PCR Detection Kit - Protocol
Contents
1. SX NORGEN BIOTEK wie CORPORATION 3430 Schmon Parkway Thorold ON Canada L2V 4Y6 Phone 866 667 4362 e 905 227 8848 Fax 905 227 1061 Email techsupport norgenbiotek com E coli 0157 H7 PCR Detection Kit Product 41300 Product Insert E coli O0157 H7 is a rod shaped gram negative bacterium It is an enterohemorrhagic strain of the common E coli bacterium and infection by the O157 H7 strain is commonly associated with hemorrhagic colitis E coli O157 H7 is recognized by its somatic cell wall antigen 0157 and its flagella antigen H7 In addition E coli O157 H7 is known to produce Shiga like toxins which cause severe symptoms While most patients can recover from the infection up to 15 of the patients may develop hemolytic uremic syndrome a type of kidney failure that could be fatal Infection of E coli O157 H7 usually results from consumption of poorly prepared food including undercooked meat particularly ground beef untreated water or raw unpasteurized milk Principle of the Test and Product Description Norgen s E coli 0157 H7 PCR Detection Kit constituents a ready to use system for the isolation and the detection of E coli O157 H7 using end point PCR The kit first allows for the isolation of bacterial DNA from milk samples or enriched samples from food using spin column chromatography based on Norgen s proprietary resin The DNA is isolated free from inhibitors and can then be used as the template in2. a PCR reaction for E coli O157 H7 detection using the provided E coli O157 H7 Master Mix The E coli O157 H7 Master Mix contains reagents and enzymes for the specific amplification of the two Shiga like toxin regions 348 bp and 587 bp of the E coli O157 H7 genome In addition Norgen s E coli 0157 H7 PCR Detection Kit contains a second Master Mix 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 Digestion Buffer 3 mL Lysis Solution 12 mL Binding Solution 4 mL Wash Solution 15 mL Wash Solution II 5 mL Elution Buffer 8 mL Proteinase K 6 mg Lysozyme 60 mg Mini 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 IsoC Isolation Control PosC Positive Control 4 The positive control is cloned E coli 0157 H7 DNA fragments b 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 37 C incubator 55 C incubator Storage C
3. lt PCR Control Figure 2 A representative 1X TAE 1 7 agarose gel showing the amplification of Isolation Control and PCR Control under different conditions using the Control 2X PCR Master Mix 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 Interpretation Positive xX X xX X Valid Control Negative i Control X Valid Sample X X X X Positive Sample X X Negative Sample X X X Positive Sample X X X Positive Sample X X Positive Negative Other Sample X X X Shiga toxin producing bacteria Negative Other Sample X X X Shiga toxin producing bacteria For results obtained that are not covered in Table 5 above please refer to the Troubleshooting Section E E coliO157 H7 PCR Assay Specificity and Sensitivity e The specificity of Norgen s E coli 0157 H7 PCR Detection Kit is first and foremost ensured by the selection of the E coli O157 H7 specific primers as well as the
4. selection of stringent reaction conditions The primers were checked for possible homologies to all 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 food borne disease causing bacteria commonly found E coli Streptococcus agalatiae Streptococcus dysgalatiae Streptococcus uberis Staphylococcus aureus Listeria monocytogenes Salmonella sp Pseudomonas aeruginosa F Linear Range e The linear range analytical measurement of Norgen s E coli O157 H7 PCR Detection Kit was determined by analysing a dilution series of a E coli O157 H7 quantification standard ranging from 1 x 107 cfu ul to 1 x 10 cfu ul e Each dilution has been tested in replicates n 4 using Norgen s E coli O157 H7 PCR Detection Kit on 1X TAE 1 7 Agarose gel e The linear range of Norgen s E coli O157 H7 PCR Detection Kit has been determined to cover concentrations from 1 x 10 cfu ul to at least 1 x 10 cfu ul e Under the conditions of the Norgen s E coli O157 H7 DNA Isolation procedure Norgen s E coli 0157 H7 PCR Detection Kit covers a linear range from 1 000 cfu mL milk to at least 1 x 107 cfu mL milk Frequently Asked Questions 1 How many samples should be included per PCR run e Norgen s E coli 0157 H7 PCR Detection Kit is designed to test 24 samples For every 6 samp
5. 0 RPM b Discard the flowthrough and reassemble 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 B E coli 0157 H7 PCR Assay Preparation Notes Before use suitable amounts of all PCR components should be completely thawed at room temperature vortexed and centrifuged briefly The amount of E coli 0157 H7 PCR Master Mix and Control 2X PCR Master Mix provided is enough for up to 32 PCR reactions 24 sample PCR 4 positive control PCR and 4 no template control PCR each For each sample one PCR reaction using the E coli O157 H7 2X PCR Master Mix and one PCR reaction using Control 2X PCR Master Mix should be set up in order to have a proper interpretation of the result For every PCR run one reaction containing E coli O157 H7 Positive Control PosC and one reaction as no template cont
6. Isolation Control soC An Isolation Control soC is supplied This allows the user to control the DNA isolation procedure For this assay add the Isolation Control IsoC to the lysate during the isolation procedure The Isolation Control soC must not be added to the sample material directly Do not freeze and thaw the Isolation Control IsoC more than 2 times The Isolation Control lsoC must be kept on ice at all times during the isolation procedure e The steps for preparing the lysate are different depending on the starting material Step 1 However the subsequent steps are the same in all cases Steps 2 4 e Milk Samples Freshly collected milk samples or enriched samples are recommended Frozen milk samples may be used but note that the sensitivity of detection may be decreased Milk samples collected into preservatives such as bronopol are not recommended e The PCR components of E coli 0157 H7 PCR Detection Kit should remain at 20 C until DNA is extracted and ready for PCR amplification 1A Lysate Preparation from Milk a Vortex the milk sample for 10 to 15 seconds or invert several times to mix b Aliquot a maximum of 1 mL of milk into a microcentrifuge tube Note Up to 1 mL of milk is recommended for normal milk samples For samples with high leukocyte counts up to 200 uL of milk sample is recommended If the sample is very viscous and difficult to pipette pass the sample through an 18 gauge
7. ach 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 with laboratory detergent and water and then with 1 v v sodium hypochlorite Protocol A E coli 0157 H7 Genomic DNA Isolation Precaution All samples must be treated as potentially infectious material Important Notes Prior to Beginning Protocol e Avariable 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 may be observed e Preheat an incubator or heating block to 37 C and another 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 Add the provided amount of Digestion Buffer to the tube containing the Lysozyme and mix well Aliquot the Digestion Buffer into small fractions and store the unused portions at 20 C until needed e Prepare a working concentration of Wash Solution Il 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
8. con will be predominant and thus the PCR control PCRC as well as the Isolation Control IlsoC 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 and Lysozyme 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 37 C or 55 C for Lysozyme and Proteinase K respectively e At other temperatures the activity of both the Proteinase K and Lysozyme 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 I forgot to add Isolation Con
9. cordance with Norgen s ISO 9001 and ISO 13485 certified Quality Management System each lot of Norgen s E coli O157 H7 PCR Detection Kit including the E coli O157 H7 2x PCR Master Mix Control 2x PCR Master Mix Isolation Control IsoC and E coli O157 H7 Positive Control PosC are tested against predetermined specifications to ensure consistent product quality Product Use Limitations Norgen s E coli 0157 H7 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 E coli O157 H7 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 The Binding Solution and Wash Solution I contain guanidine salts and should be handled with care Guanidine salts form highly reactive compounds when combined with ble
10. gen Biotek Corp P141300 3
11. les a non 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 E coli O157 H7 targets 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 E coli 0157 H7 negative 5 How should it be interpreted if only the E coli O157 H7 targets and the PCR control PCRC were amplified in a sample e The sample tested can be considered as E coli 0157 H7 positive 6 How should it be interpreted if only the E coli 0157 H7 targets were amplified in a sample e The sample tested should be considered as E coli 0157 H7 positive At high E coli O157 H7 cell input the E coli O157 H7 ampli
12. onditions and Product Stability All buffers 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 Lysozyme should be stored at 20 C upon arrival and the Digestion Buffer should be stored at 20 C after addition of the Lysozyme 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 E coli 0157 H7 2x PCR Master Mix Control 2x PCR Master Mix Isolation Control IsoC and E coli O157 H7 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 ac
13. ramming 1 Program the thermocylcer according to the program shown in Table 4 below 2 Run PCR Table 4 E coli 0157 H7 Assay Program One Step PCR Cycle Step Temperature Duration Cycle 1 Step 1 95 C 5 min Step 1 94 C 15 sec Cycle 2 35x 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 o0 D E coli O0157 H7 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 E coli 0157 H7 detection and one set of PCR for controls detection 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 Sample results are provided below M EcoliO157 H7 NC 2000 1500 1000 750 500 E coli O157 H7 STX2 Target e E coli 0157 H7 STX1 Target 150 Figure 1 A representative 1X TAE 1 7 agarose gel showing the amplification of E coli 0157 H7 using the E coli 0157 H7 2X PCR Master Mix The size of the E coli 0157 H7 target amplicon corresponds to 348 bp STX1 and 587 bp STX2 as represented by the provided DNA Marker M NC Negative Control 1 2 3 4 5 6 NC 2000 1500 1000 750 500 300 Isolation Control 150
14. rol must be included for proper interpretation of results e The recommended minimum number of DNA samples tested per PCR run is 6 Using a lower volume from the sample than recommended may affect the sensitivity of E coli 0157 H7 Limit of Detection 1 Prepare the PCR for sample detection Set 1 using E coli O157 H7 2X PCR Master Mix and control detection Set 2 using Conrtol 2X PCR Master Mix 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 E coli O157 H7 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 pL Total Volume 20 uL Therefore at a minimum each PCR run will contain 6 separate PCR reactions C PCR Assay Prog
15. s apop Note Ensure that the provided Lysozyme has been added to the Digestion Buffer f After incubation add 300 uL of Lysis Solution and 10 uL of reconstituted Proteinase K to the digestion mixture and mix well by vortexing g 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 Isolation Control IsoC and 180 uL of 96 100 ethanol to the lysis mixture and mix by vortexing Note Ensure that the Isolation Contro soC is added for subsequent control detection in the PCR protocol b For Milk only Spin the sample for 10 seconds at 14 000 x g 14 000 RPM A thin layer of lipid may form on the top of the aqueous phase Using a pipette carefully transfer the clear aqueous phase only to a spin column that has been attached to a collection tube For other samples Transfer the lysate ethanol mix to a spin column that has been attached to a collection tube c 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 00
16. syringe a few times to reduce the viscosity c Centrifuge at 14 000 x g 14 000 RPM for 3 minutes d Pour off the supernatant by quickly inverting the tube and gently tapping it against the wall of the waste container This tapping is to ensure that the creamy layer present on the top of the milk sample after centrifugation is removed Clean any remaining white solid from the microcentrifuge tube wall by using a provided clean cotton swab Ensure that the pellet is not dislodged e Resuspend the pellet in 100 uL of Digestion Buffer Incubate at 37 C for 45 minutes Note Ensure that the provided Lysozyme has been added to the Digestion Buffer f After incubation add 300 uL of Lysis Solution and 10 uL of reconstituted Proteinase K to the digestion mixture and mix well by vortexing g Incubate the lysate at 55 C for 45 minutes Mix the lysate occasionally by vortexing 1B Lysate Preparation from Enriched or Cultured Samples a Grow the sample under standard protocol for E coli 0157 H7 Note Standard culture of E coli 0157 H7 involves dilution of food sample at a 1 10 ratio in Tryptic Soy Broth supplemented with Novobiocin and grow at 42 C for 22 to 24 hours Transfer up to 1 mL of enriched culture to a nuclease free microcentrifuge tube Centrifuge at 14 000 x g 14 000 RPM for 3 minutes Pour off the supernatant by quickly inverting the tube Resuspend the pellet in 100 uL of Digestion Buffer Incubate at 37 C for 45 minute
17. trol IsoC during the Isolation e It is recommended that the isolation is repeated Reference Perna NT et al 2001 Genome sequence of enterohaemorrhagic Escherichia coli 0157 H7 Nature 409 529 533 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 products 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 Schmon Parkway Thorold ON Canada L2V 4Y6 Phone 905 227 8848 Fax 905 227 1061 Toll Free in North America 1 866 667 4362 2013 Nor
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