Listeria Monocytogenes PCR Detection Kit
Contents
1. 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 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 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 Holko J Urbanova M Kantikova K Pastorova V Kmee 2002 PCR Detection of Listeria monocytogenes in Mi2. 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 E Listeria monocytogenes PCR Assay Results Interpretation 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 L monocytogenes 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 L monocytogenes NC C 2000 1500 1000 750 500 300 L monocytogenes Target 150 Figure 1 A representative 1X TAE 1 7 agarose gel showing the amplification of L monocytogenes L monocytogenes Target using the L monocytogenes 2x PCR Master Mix The size of the L monocytogenes target amplicon corresponds to 366 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 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
3. 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 L monocytogenes 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 L monocytogenes negative 5 How should it be interpreted if only the L monocytogenes target and the PCR control PCRC were amplified in a sample e The sample tested can be considered as L monocytogenes positive 6 How should it be interpreted if only the L monocytogenes target was amplified in a sample e The sample tested should be considered as L monocytogenes positive At high L monocytogenes cell input the L monocytogenes 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
4. the bottle has a box that can be checked to indicate that ethanol has been added e Isolation Control soC A 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 Controk 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 IsoC must be kept on ice at all times during the isolation procedure e Bacterial Samples Immediate uses of the enriched samples are recommended If the sample 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 Listeria monocytogenes PCR Detection Kit should remain at 20 C until DNA is extracted and ready for PCR amplification 1 Lysate Preparation Vortex the enriched bacteria sample for 10 to 15 seconds or invert several times to mix Aliquot 1 mL of enriched bacteria 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 100 uL of Digestion Buffer Incubate at 37 C for 45 minutes o2009 Note Ensure that the provided Lysozyme has been added to the Digestion
5. 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 reaction Control Reaction Interpretation Listeria Listeria Listeria monocytogenes monocytogenes monocytogenes Target Band IsoC Band PCRC Band 366 bp 499 bp 150 bp Positive X X X Valid Control Negative Control ee 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 Listeria monocytogenes PCR Assay Specificity and Sensitivity e The specificity of Norgen s Listeria monocytogenes PCR Detection Kit is first and foremost ensured by the selection of the L monocytogenes specific primers as well as the 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 th
6. 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 Control soC is added for subsequent control detection in the PCR protocol b Using a pipette carefully transfer the lysate with ethanol 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 14 000 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 a
7. E gt x NORGEN BIOTEK wi CORPORATION Listeria Monocytogenes PCR Detection Kit Meat Product Product 32000 3430 Schmon Parkway Thorold ON Canada L2V 4Y6 Phone 866 667 4362 e 905 227 8848 Fax 905 227 1061 Email techsupport norgenbiotek com Product Insert Listeria monocytogenes have emerged as significant foodborne pathogens that pose a serious public health problem As the causative agent of Listeriosis L monocytogenes has the highest rate of fatality rate among all foodborne pathogens L monocytogenes is a facultatively intracellular Gram positive bacterium Due to its ability to survive high and low temperatures as well as low pH it could resist various food processing technologies as well as to grow at storage temperature L monocytogenes is know to be associated with raw meat unpasteurized milk and dairy products vegetables and seafood As little as 1000 organisms may cause the disease with pregnant new born and immunocompromised individuals the most susceptible Principle of the Test and Product Description Norgen s Listeria monocytogenes PCR Detection Kit constituents a ready to use system for the isolation the detection of L monocytogenes 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 and can then be used as t
8. genbiotek 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 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 Listeria monocytogenes Enrichment Important Notes Prior to Use e The followings provide a protocol for preparing 1 L of selective media for L monocytogenes enrichment enough for a total of 4 samples Each 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 e The Listeria monocytogenes Supplement for Selective Enrichment is provided in tubes of 105 mg of powder Enough for 4 enrichments To reconstitute each tube add 1 mL of Sterile Water All reconstituted materials should be used immediately 1 Listeria monocytogenes Enrichment Broth Preparation Dissolve 36 g of L monocytogenes Enrichment Media in 1 L of distilled water Ensure a final pH at 7 3 0 2 Autoclave at 121 C for 15 minutes Allow the broth to cool to 30 C or below Add 1 mL of reconstituted L monocytogenes Supp
9. he template in a PCR reaction for L monocytogenes detection using the provided L monocytogenes 2x PCR Master Mix The L monocytogenes Master Mix contains reagents and enzymes for the specific amplification of a 366 bp region of the L monocytogenes genome In addition Norgen s Listeria monocytogenes PCR Detection Kit contains a second Master Mix the Control 2x PCR 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 L monocytogenes Enrichment Media Contents 216g L monocytogenes Supplement for Selective Enrichment 630 mg Digestion Buffer 3 mL Lysis Solution 12 mL Binding Solution Wash Solution 4 mL 15 mL Wash Solution II 5 mL Elution Buffer Proteinase K 8 mL 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 The positive control is purified L monocytogenes genomic DNA fragments The isolation control is a cloned PCR product Customer Supplied Reagents and Equipment e Disposable powder free glo
10. lement for Selective Enrichment Mix well by swirling oQ0op 2 Listeria monocytogenes 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 L monocytogenes Enrichment Broth prepared in A 1 Incubate at 30 C for 24 hours Proceed to Listeria monocytogenes Genomic DNA Isolation aop B Listeria monocytogenes 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
11. lk and Milk Products and Differentiation of Suspect Isolates ACTA VET BRNO 71 125 131 H A Bassler S J A Flood K J Livak J Marmaro R Knorr and C A Batt 1995 Use of a Fluorogenic Probe in a PCR Based Assay for the Detection of Listeria monocytogenes App Env Microl 61 3724 3728 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 norge
12. nbiotek 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 P132000 3
13. nd 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 Listeria monocytogenes 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 L monocytogenes 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 L monocytogenes 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 L monocytogenes Positive Control L monocytogenes 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 L monocytogenes Limit of Detection 1 Prepare the PCR for sample detection Set 1 usi
14. ng L monocytogenes 2x PCR Master Mix and control detection Set 2 using Control 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 L monocytogenes 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 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 Total Volume Volume Per RT PCR Reaction 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 Nuclease Free Water Volume Per PCR Reaction 10 pL Total Volume 20 uL Therefore at a minimum each PCR run will contain 6 separate PCR reactions D PCR Assay Programming 1 Program the thermocylcer according to the program shown in Table 4 below 2 Run PCR Table 4 L monocytogenes 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
15. nts 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 Listeria monocytogenes PCR Detection Kit including the L monocytogenes 2x PCR Master Mix Control 2x PCR Master Mix Isolation Control IsoC and L monocytogenes Positive Control PosC are tested against predetermined specifications to ensure consistent product quality Product Use Limitations Norgen s L monocytogenes 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 Listeria monocytogenes 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 nor
16. us 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 Salmonella sp G Linear Range e The linear range analytical measurement of Norgen s Listeria monocytogenes PCR Detection Kit was determined by analysing a dilution series of a L monocytogenes quantification standard ranging from 1 x 10 cfu ul to 1 x 10 cfu l e Each dilution has been tested in replicates n 4 using Norgen s Listeria monocytogenes PCR Detection Kit on 1X TAE 1 7 Agarose gel e The linear range of Norgen s Listeria monocytogenes 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 Listeria monocytogenes DNA Isolation procedure Norgen s Listeria monocytogenes 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 Listeria monocytogenes 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
17. ves 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 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 L monocytogenes 2x PCR Master Mix Control 2x PCR Master Mix Isolation Control IsoC and L monocytogenes 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 compone
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