Campylobacter jejuni PCR Detection Kit
Contents
1. 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 C jejuni 2x PCR Master Mix Control 2X PCR Master Mix Isolation Control lsoC and C jejuni 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 Campylobacter jej2. been tested in replicates n 4 using Norgen s Campylobacter jejuni PCR Detection Kit on 1X TAE 1 7 Agarose gel The linear range of Norgen s Campylobacter jejuni PCR Detection Kit has been determined to cover concentrations from 1 x 10 cfu ul to at least 1 x 10 cfu ul Under the conditions of the Norgen s Campylobacter jejuni DNA Isolation procedure Norgen s Campylobacter jejuni PCR detection Kit covers a linear range from 1 000 cfu mL milk to at least 1 x 10 cfu mL milk Frequently Asked Questions 1 How many samples should be included per PCR run e Norgen s Campylobacter jejuni PCR Detection Kit is designed to test 24 samples For every 6 samples 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 C jejuni target nor the Isolation Control IsoC amplified
3. 0 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 minutes 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 o 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 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 Spi
4. CR 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 C jejuni NC lt lt C jejuni Target Figure 1 A representative 1X TAE 1 7 agarose gel showing the amplification of C jejuni under different concentration C jejuni Target using the C jejuni 2X PCR Master Mix The size of the C jejuni target amplicon corresponds to 321 bp 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 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 Ta
5. Fax 905 227 1061 BIOTEK si CORPORATION Email techsupport norgenbiotek com abl 3430 Schmon Parkway lt Thorold ON Canada L2V 4Y6 x Phone 866 667 4362 e 905 227 8848 Campylobacter jejuni PCR Detection Kit Product Insert Product 36100 Campylobacter jejuni is a curved rod shaped and microaerophillic gram negative bacterium It is one of the most common causal agents of gastroenteritis with diarrhea as the main symptom While infection of C jejuni is seldom life threatening it is considered one of the most common food borne bacteria with over 2 million people infected per year in US alone Infection of C jejuni usually results from consumption of poorly prepared food including undercooked meat particularly poultry untreated water or raw unpasteurized milk Traditional identification of C jejuni involves culturing however the microaerophilic characteristic of this bacterium make the enrichment process laborious and costly Principle of the Test and Product Description Norgen s Campylobacter jejuni PCR Detection Kit constituents a ready to use system for the isolation and the detection of C jejuni using end point PCR The kit first allows for the isolation of bacterial DNA from milk samples poultry carcass rinse or enriched samples 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 in a PCR reaction for C jejuni detection using
6. ed 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 1C Lysate Preparation from Enriched or Cultured Samples a Grow the sample under standard protocol for C jejuni Note Standard culture of C jejuniinvolves growth on Campy Cefex agar under a microaerobic environment at 42 C for 36 to 48 hours b Scrap cells off the plate and resuspend in 1 mL of sterile water or sterile saline 0 9 NaCl Transfer the resuspension 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 e Resuspend the pellet in 100 uL of Digestion Buffer Incubate at 37 C for 45 minutes a9 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 soC 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 or Carcass Rinse only Spin the sample for 10 seconds at 14 00
7. 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 C jejuni negative 5 How should it be interpreted if only the C jejuni target and the PCR control PCRC were amplified in a sample e The sample tested can be considered as C jejuni positive 6 How should it be interpreted if only the C jejuni target was amplified in a sample e The sample tested should be considered as C jejuni positive At high C jejuni cell input the C jejuni amplicon will be predominant and thus the PCR control 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 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 act
8. he 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 uL 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 C PCR Assay Programming 1 Program the thermocylcer according to the program shown in Table 4 below 2 Run PCR Table 4 C jejuni 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 oo D Campylobacter jejuni 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 C jejuni detection and one set of P
9. ivity 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 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 Related Products Product Milk Bacterial DNA Isolation Kit 21500 Bacterial Genomic DNA Isolation Kit 17900 Reference J Parkhill et al 2000 The genome sequence of the food borne pathogen Campylobacter jejuni reveals hypervariable sequences Nature 403 665 668 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 Campylobacter jejuni PCR Detection Kit or NORGEN products in general please do not hesitate to contact us NORGEN customers are a valuable source of information regarding adva
10. n for an additional 2 minutes at 14 000 x g 14 000 RPM to complete the DNA elution B Campylobacter jejuni PCR Assay Preparation Notes e Before use suitable amounts of all PCR components should be completely thawed at room temperature vortexed and centrifuged briefly The amount of C jejuni 2X 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 C jejuni 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 C jejuni Positive Control PosC and one reaction as no template control 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 C jejuni Limit of Detection 1 Prepare the PCR for sample detection Set 1 using C jejuni 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 C jejuni detection reaction and one control reaction is prepared for each DNA sample Adjust the final volume of t
11. nced 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 Norgen Biotek Corp PI36100 4
12. our 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 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 Poultry Carcass or Meat Rinse a Collect poultry carcass or meat rinse by standard procedure Note For example place the carcass or meat sample in a plastic bag containing 100 mL of sterile water Seal the bag and shake for 3 minutes Aliquot a maximum of 1 mL of the rinse into a 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 minutes 9205 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 reconstitut
13. re For this assay add the Isolation Control IsoC to the lysate during the isolation procedure The Isolation Controk soC must not be added to the sample material directly Do not freeze and thaw the C jejuni 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 Campylobacter jejuni 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 syringe a few times to reduce the viscosity c Centrifuge at 14 000 x g 14 000 RPM for 3 minutes d P
14. rget reaction Control Reaction Interpretation C jejuni Target C jejuni lsoC C jejuni PCRC Band 321 bp Band 499 bp Band 150 bp Positive Control X X X Valid Negative Control x vaig 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 E campylonectet jejuni PCR Assay Specificity and Sensitivity The specificity of Norgen s Campylobacter jejuni PCR Detection Kit is first and foremost ensured by the selection of the C jejuni specific primers as well as the 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 Ecoli Streptococcus agalatiae Streptococcus dysgalatiae Staphylococcus aureus Listeria monocytogenes Salmonella sp F Linear Range The linear range analytical measurement of Norgen s Campylobacter jejuni PCR Detection Kit was determined by analysing a dilution series of a C jejuni quantification standard ranging from 1 x 10 cfu ul to 1 x 10 cfu l Each dilution has
15. 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 Campylobacter jejuni 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 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 Isolation Control soC An Isolation Control soC is supplied This allows the user to control the DNA isolation procedu
16. the provided C jejuni Master Mix C jejuni Master Mix contains reagents and enzymes for the specific amplification of a 321 bp region of the C jejuni genome In addition Norgen s Campylobacter jejuni 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 IsoC 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 4mL Wash Solution 15 mL Wash Solution II 5 mL Elution Buffer 8 mL Proteinase K 6 mg Lysozyme 60 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 cloned C jejuni 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 37 C incubator 55 C incubator Storage Conditions and Product Stability All buffers should be kept tightly sealed and stored at room temperature 15 25
17. uni PCR Detection Kit including the C jejuni 2x PCR Master Mix Control 2X PCR Master Mix Isolation Control IsoC and C jejuni Positive Control PosC are tested against predetermined specifications to ensure consistent product quality Product Use Limitations Norgen s C jejuni 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 Campylobacter jejuni 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 bleach thus care must be taken to properly dispose of any of these solutions If liquid containing these buffers is spilt clean with
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