Clostridium difficile PCR Detection Kit - Protocol
Contents
1. Detection Kit should remain at 20 C until DNA is extracted and ready for PCR amplification 1 Lysate Preparation a Add up to 200 mg of stool sample to a provided Bead Tube and add 1 mL of Lysis Solution Vortex briefly to mix stool and Lysis Solution b Secure tube horizontally on a flat bed vortex pad with tape or secure the tube in any commercially available bead beater equipment e g Scientific Industries Disruptor Genie Vortex for 3 minute at maximum speed c Centrifuge the tube for 3 minutes at 14000 x g 14 000 RPM d Transfer up to 600 uL of supernatant to a DNAase free microcentrifuge tube not provided e Add 100 uL of Binding Solution mix by inverting the tube a few times and incubate for 10 minutes on ice f Spin the lysate for 3 minutes to pellet any cell debris g Using a pipette transfer up to 650 uL of supernatant avoid contacting the pellet with the pipette tip into a 2 mL DNAase free microcentrifuge tube not provided h Add 10 uL of Isolation Control soC 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 Add an equal volume of 70 ethanol provided by the user to the lysate collected above 100 uL of ethanol is added to every 100 uL of lysate Vortex to mix Proceed to Step 2 2 Binding to Column a b C Assemble a spin column with one of the provided collection tubes Ap2. and 5 uL of sample DNA may be used as template Ensure that one C difficile 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 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 difficile 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 eo D Clostridium difficile PCR Assay Results Interpretation 1 For the analysis of the PCR data the entire 15 20 uL PCR Reaction should
3. Notes Prior to Beginning Protocol e All centrifugation steps are carried out in a benchtop microcentrifuge at 14 000 x g 14 000 RPM except where noted All centrifugation steps are performed at room temperature 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 Ensure that all solutions are at room temperature prior to use e Prepare a working concentration of Wash Solution by adding 25 mL of 95 100 ethanol provided by the user to the supplied bottle containing the concentrated Wash Solution This will give a final volume of 36 mL The label on the bottle has a box that may be checked to indicate that the ethanol has been added e The maximum input of fresh or frozen stool sample is 200 mg If the stool sample is in a e suspension an equivalent volume should be processed e 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 soC must be kept on ice at all times during the isolation procedure e The PCR components of the Clostridium difficile PCR
4. 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 difficile 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 2000 1500 1000 500 300 150 C difficile NC _ C difficile Target Figure 1 A representative 1X TAE 1 7 agarose gel showing the amplification of C difficile C difficile Target using the 2X C difficile PCR Master Mix The size of the C difficile target amplicon corresponds to 325 bp as represented by the provided DNA Marker M NC Negative Control Isolation control gt PCR 3 control Figure 2 A representative 1X TAE 1 5 agarose gel showing the amplification of Isolation Control and PCR Control under different conditions using the Control 2X PCR 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 and 2 showed detection of both Isolation Control and PCR Control suggesting that the DNA isolation as well as the PCR reaction was successful Lane 3 and 4 showed only the detection of PCR Control suggesting that while the PCR was successful the isolation failed to recove
5. difficile detection using the provided C difficile Master Mix The C difficile Master Mix contains reagents and enzymes for the specific amplification of a 325 bp region of the C difficile genome In addition Norgen s Clostridium difficile 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 PCR control PCRC or Isolation Control IsoC respectively This kit is designed to allow for the testing of 24 samples Kit Components Component Contents Lysis Solution 30 mL Binding Solution 3 mL Wash Solution 11 mL Elution Buffer 3 mL Bead Tube 24 Mini Spin Columns 24 Collection Tubes 24 Elution tubes 1 7 mL 24 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 difficile 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 Flat bed vortex or bead beater equipment 95 100 ethanol 70 ethanol 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 w
6. e has not been eluted spin the column at 14 000 x g 14 000 RPM for 2 additional minutes 5 Storage of DNA The purified genomic DNA can be stored at 2 8 C for a few days For longer term storage 20 C is recommended B Clostridium difficile 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 C difficile 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 e For each sample one PCR reaction using the C difficile 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 e For every PCR run one reaction containing C difficile Positive Control C difficile 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 e Using a lower volume from the sample than recommended may affect the sensitivity of C difficile Limit of Detection 1 Prepare the PCR for sample detection Set 1 using C difficile 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
7. g gt x NORGEN BIOTEK wi 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 Clostridium difficile PCR Detection Kit Product 37100 Product Insert Clostridium difficile is rod shaped gram positive bacterium It is the main causal agent of antibiotic associated diarrhea and pseudomembranous colitis The colonization of intestines by C difficile is usually associated with the elimination of natural intestinal flora as a result of antibiotic application and is frequently reported in health care centers While C difficile infection could be severe and life threatening particularly among the elderly many patients are asymptomatic making diagnosis challenging during outbreaks The tradition method of detecting C difficile infection is by cytotoxicity test of the toxin produced by the bacterium but such protocols usually require extensive time before conclusion can be made Principle of the Test Norgen s Clostridium difficile PCR Detection Kit constituents a ready to use system for the isolation without enrichment and the detection of C difficile using end point PCR The kit first allows for the isolation of bacterial DNA from patient s stool sample 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
8. icrobiology 41 730 734 Related Products Product Stool DNA Isolation Kit 27600 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 Clostridium difficile PCR Detection Kit 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 Schmon 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 P137100 3
9. ithout showing any reduction in performance The C difficile 2X PCR Master Mix Control 2X PCR Master Mix Isolation Control IsoC and C difficile 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 Clostridium difficile PCR Detection Kit including the C difficile 2X PCR Master Mix Control 2X PCR Master Mix Isolation Control IsoC and C difficile Positive Control PosC are tested against predetermined specifications to ensure consistent product quality Product Use Limitations Norgen s C difficile PCR Detection Kit is designed for research purposes
10. 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 difficile negative 5 How should it be interpreted if only the C difficile target and the PCR control PCRC were amplified in a sample e The sample tested can be considered as C difficile positive 6 How should it be interpreted if only the C difficile target was amplified in a sample e The sample tested should be considered as C difficile positive At high C difficile cell input the C difficile 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 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 9 What If I forgot to add Isolation Control IsoC during the Isolation e It is recommended that the isolation is repeated Reference B langer SD Boissinot M Clairoux N Picard FJ and Bergeron MG 2003 Rapid Detection of Clostridium difficile in Feces by Real Time PCR Journal of Clinical M
11. n series of a C difficile quantification standard ranging from 1 x 10 cfu ul to 1 x 107 cfu ul e Each dilution has been tested in replicates n 4 using Norgen s Clostridium difficile PCR Detection Kit on 1X TAE 1 7 Agarose gel e The linear range of Norgen s Clostridium difficile PCR Detection Kit has been determined to cover concentrations from 1 x 10 cfu ul to at least 1 x 10 cfu ul of isolated DNA Frequently Asked Questions 1 How many samples should be included per PCR run e Norgen s Clostridium difficile PCR Detection Kitis 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 difficile target nor the Isolation Control IsoC amplified for a sample e This indicates a poor isolation The isolation procedure
12. 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 Clostridium difficile 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 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 Clostridium difficile Genomic DNA Isolation Precaution All samples must be treated as potentially infectious material Important
13. ply 650 uL of the clarified lysate with ethanol onto the column and centrifuge for 1 minute at 14000 x g 14 000 RPM Discard the flowthrough and reassemble the spin column with the collection tube Note Ensure the entire lysate volume has passed through into the collection tube by inspecting the column If the entire lysate volume has not passed spin for an additional minute Repeat step 2b with the remaining volume of lysate mixture 3 Column Wash a Apply 400 uL of Wash Solution to the column and centrifuge for 1 minute Note Ensure the entire wash solution has passed through into the collection tube by inspecting the column If the entire wash volume has not passed spin for an additional minute b Discard the flowthrough and reassemble the spin column with its collection tube c Repeat steps 3a and 3b to wash column a second time d Wash column a third time by adding another 400 uL of Wash Solution and centrifuging for 1 minute e Discard the flowthrough and reassemble the spin column with its collection tube f Spin the column for 2 minutes in order to thoroughly dry the resin Discard the collection tube 4 DNA Elution a b c Place the column into a fresh 1 7 mL Elution tube provided with the kit Add 75 uL of Elution Buffer to the column Centrifuge for 2 minutes at 200 x g 2 000 RPM followed by a 2 minute spin at 14 000 x g 14 000 RPM Note the volume eluted from the column If the entire volum
14. r even the spiked in Isolation control Table 5 Interpretation of PCR Assay Results Input Type Target Control Reaction Interpretation reaction C difficile IsoC Band C difficile Target Band 499 bp PCRC Band 324 bp 150 bp Positive xX xX X Valid Control Negative j 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 E Clostridium difficile PCR Assay Specificity and Sensitivity e The specificity of Norgen s Clostridium difficile PCR Detection Kit is first and foremost ensured by the selection of the C difficile 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 commonly found bacteria Ecoli Listeria monocytogenes Streptococcus agalatiae Streptococcus dysgalatiae Staphylococcus aureus Salmonella sp F Linear Range e The linear range analytical measurement of Norgen s Clostridium difficile PCR Detection Kit was determined by analysing a dilutio
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