Leptospira interrogans PCR Detection Kit
Contents
1. please refer to the Troubleshooting Section E Leptospira PCR Assay Specificity and Sensitivity e The specificity of Norgen s Leptospira interrogans PCR Detection Kit is first and foremost ensured by the selection of the Leptospira specific primers as well as the selection of stringent reaction conditions The Leptospira specific primers were checked for possible homologies to GenBank published sequences by sequence comparison analysis and published Leptospira strains F Linear Range e The linear range of Norgen s Leptospira interrogans PCR Detection Kit was determined by analysing a dilution series of a Leptospira quantification standards ranging from 1pg to 10 ng e Each dilution has been tested in replicates n 4 using Norgen s Leptospira interrogans PCR Detection Kit on a 1X TAE 1 7 agarose gel e The linear range of Norgen s Leptospira interrogans PCR Detection Kit has been determined to cover concentrations from 1pg to 10 ng e Under the conditions of the Norgen s Leptospira DNA Isolation procedure Norgen s Leptospira interrogans PCR Detection Kit covers a linear range from 100 copies to 1 x 10 copies Frequently Asked Questions 1 How many samples should be included per PCR run e Norgen s Leptospira interrogans PCR Detection Kit is designed to test 24 samples For every 6 samples a non template control Nuclease Free Water and a Positive Control must be included It is preferable to pool and test 6 s2. 00 RPM followed by 1 minute at 14 000 RPM B Leptospira 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 LEP 2x PCR Master Mix and Control 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 For each sample one PCR reaction using the LEP 2x PCR Mastermix and one PCR reaction using Control 2x PCR Mastermix should be set up in order to have a proper interpretation of the result For every PCR run one reaction containing LEP 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 e Using a lower volume from the sample than recommended may affect the sensitivity of Leptospira interrogans Limit of Detection 1 Prepare the PCR for sample detection Set 1 using LEP 2x PCR Mastermix and control detection Set 2 using Control 2x PCR 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 LEP 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
3. 3430 Schmon Parkway Thorold ON Canada L2V 4Y6 Phone 866 667 4362 e 905 227 8848 Fax 905 227 1061 Email techsupport norgenbiotek com NORGEN BIOTEK wi CORPORATION ay gt lt Leptospira interrogans PCR Detection Kit Product Insert Product 44600 Pathogen Information Leptospirosis is a disease of both humans and animals The causal agent of the disease is the bacterium Leptospira interrogans Leptospirosis is transmitted via contact with infected urine venereal and placental transfer bite wounds or ingestion Indirect transmission may also occur through contaminated food water or bedding After infection the bacteria penetrate mucous membranes and multiply rapidly upon entering the blood system spreading to other tissues including the kidneys liver spleen nervous system eyes and genital tract Symptoms of acute infections include fever shivering muscle tenderness vomiting and rapid dehydration The disease is treatable with antibiotics fluid replacement and anti emetic drugs as well as by controlling problems associated with the corresponding kidney and liver functions Early rapid diagnosis of this disease is essential to treat the illness before any serious complications arise Principle of the Test Norgen s Leptospira interrogans PCR Detection Kit constituents a ready to use system for the isolation and detection of Leptospira interrogans using end point PCR The kit first allows for the isolation of Leptosp
4. Figure 1 A representative 1X TAE 1 5 agarose gel showing the amplification of Leptospira at different concentrations Leptospira Target The size of the Leptospira target amplicon corresponds to 350 bp as represented by the provided DNA Marker M NTC Negative Control lt Isolation Control PCR 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 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 NTC Negative Control Table 5 Interpretation of PCR Assay Results Input Type Target Control Reaction Interpretation reaction Leptospira IsoC Band PCRC Band Target Band 499 bp 150 bp 350 bp oe x x x valid a x Valid 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
5. 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 every PCR set 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 every PCR set 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 C Leptospira PCR Assay Programming 1 Program the thermocylcer according to the program shown in Table 4 below 2 Run one step PCR Table 4 Leptospira PCR Assay Program PCR Cycle Step Temperature Duration Cycle 1 Step 1 95 C 3 min Step 1 94 C 15 sec Cycle 2 35x Step 2 58 C 15 sec Step 3 72 C 30 sec Cycle 3 Step 1 72 C 5 min Cycle 4 Step 1 4 C o D Leptospira PCR Assay Results Interpretation 1 For the analysis of the PCR data the entire 20 uL PCR Reaction should be loaded on a 1X TAE 1 5 Agarose DNA gel along with 10 uL of Norgen s DNA Marker provided 2 The PCR products should be resolved on the 1X TAE 1 5 Agarose gel at 150V for 30 minutes Gel running time will be vary depending on an electrophoresis apparatus 3 Sample results are provided below Leptospira 350bp
6. act 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 2010 Norgen Biotek Corp P144600 3
7. amples at a time If not the provided Positive Control is enough to run 3 samples at a time 2 How can I interpret my results if neither the PCR control nor the Isolation Control soC amplifies e f neither the PCR control nor the Isolation Control soC 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 showed amplification but neither the Leptospira target nor the Isolation control 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 soC was amplified in a sample e The sample tested can be considered as Leptospira negative 5 How should it be interpreted if the PCR control and the Leptospira target showed amplification in a sample e The sample tested can be considered positive It could happen when too much template was added to the reaction 6 How should it be interpreted if only the Leptospira target and the PCR control were amplified in a sample e The sample tested can be considered as Leptospira positive 7 How should it be interpreted if only the Leptospira target was amplified in a sample e It is recommended that the isolation is repeated 8 Ho
8. e pipette tips with filters PCR tubes Lysozyme 96 100 ethanol 60 C incubator 15 mL tubes Storage Conditions and Product Stability All buffers should be kept tightly sealed and stored at room temperature 15 25 C for up to 1 year without showing any reduction in performance The LEP 2x PCR Master Mix Control 2x PCR Master Mix Isolation Control IsoC and the LEP Positive Control PosC should be kept tightly sealed and stored at 20 C for up to 1 year without showing any reduction in performance Repeated thawing and freezing gt 2 x 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 Leptospira interrogans PCR Detection Kit including the LEP 2x PCR Master Mix Control 2x PCR Master Mix Isolation Control and LEP Positive Control are tested against pr
9. edetermined specifications to ensure consistent product quality Product Use Limitations Norgen s Leptospira interrogans PCR Detection Kit is designed for research purposes only 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 Disclaimers The Solution B contains guanidine hydrochloride and should be handled with care Guanidine hydrochloride forms highly reactive compounds when combined with bleach thus care must be taken to properly dispose of any of these solutions If liquid containing these solutions 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 Safety Information 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 Protocol Important Notes Prior to Beginning Protocol 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 t
10. emperature A variable speed microcentrifuge 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 First time users should read the entire manual before proceeding with the protocol Do not spin down or filter the urine sample before proceeding with the isolation as this could decrease the DNA yield Ensure that all solutions are at room temperature prior to use and that no precipitates have formed If necessary warm the solutions and mix well until the solutions become clear again Prepare a working concentration of Wash Solution by adding 21 mL of 96 100 ethanol provided by the user respectively to the supplied bottle containing the concentrated Wash Solution This will give a final volume of 30 mL The label on the bottle has a box that can be checked to indicate that Isopropanol has been added Preheat an incubator or heating block to 60 C Prepare a 400 mg mL stock solution approximately 1 7 x107 units mL of lysozyme as per supplier s instructions 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 soC to the lysate during the isolation procedure The Isolation Control IsoC must not be added to the sample material directly Do not freeze and thaw the Isolation Control soC more than 2 times The I
11. ira interrogans DNA from the urine samples using spin column chromatography based on Norgen s proprietary resin The Leptospira interrogans DNA is isolated free from inhibitors and can then be used as the template in a PCR reaction for Leptospira interrogans detection using the provided Leptospira interrogans Master Mix The Leptospira interrogans Mastermix contains reagents and enzymes for the specific amplification of a 350 region of the genome In addition Norgen s Leptospira interrogans PCR Detection contains a second Mastermix 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 Isolation Control lsoC This kit is designed to allow for the testing of 24 samples Kit Components Component Conients Solution A 10 mL Solution B 15 mL Wash Solution 9 mL Elution Buffer 3 mL Mini Filter 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 IsoC Isolation Control PosC Positive Control The isolation control is a cloned PCR product The positive control is gnomic DNA of Leptospira interrogans Customer Supplied Reagents and Equipment e Disposable powder free gloves Centrifuge with a swinging bucket rotor capable of 2000 RPM Benchtop microcentrifuge Micropipettors Steril
12. solation Control IsoC must be kept on ice at all times during the isolation procedure The PCR components of the Leptospira interrogans PCR Detection Kit should remain at 20 C until DNA is extracted and ready for PCR amplification It is important to work quickly during this procedure Specimen Collection and Sample Storage Midstream urine samples should be collected as the first flow of urine has been shown to have a higher rate of contamination Morimoto et al 2003 It is highly recommended that urine samples be collected using Norgen s Urine Collection and Preservation Tubes Cat 18111 The urine samples can be stored for at least one year at room temperature when collected directly using Norgen s Urine Collection and Preservation Tubes Alternatively urine samples collected using any other collection and preservation systems or reagents are also compatible with this kit Sample Transport Sample material should be transported in a shatterproof leak proof transport container as a matter of principle Thus a potential danger of infection due to a leakage of sample can be avoided The samples should be transported following the local and national instructions for the transport of pathogen material A Isolation of DNA 1 Add 300 uL of Solution A to 10 mL urine sample Mix well by vortexing for 10 seconds Note 1 Solution A must be mixed well before every pipetting 2 Centrifuge for 5 minutes at 2 000 RPM then discard
13. the supernatant carefully in order not to dislodge the precipitated slurry pellet 3 Add 20 uL of the previously prepared lysozyme to the precipitated slurry pellet Vortex for 10 seconds Incubate the mixture at 60 C for 20 minutes 4 Add 500 uL Solution B to the precipitated slurry pellet mix well by vortexing for 10 seconds 5 Add 10 uL Isolation Control IsoC to the mixture from Step 4 6 Add 500 uL of 96 100 Ethanol to the mix from Step 5 mix well by vortexing for 10 seconds 7 Transfer 650 uL from the previous mix into a Mini Filter Spin column and centrifuge for 1 minute at 14 000 RPM Discard the flowthrough and reassemble the spin column with its collection tube 8 Repeat Step 7 until the entire mixture from Step 6 has been transferred to the Mini Filter Spin Column 9 Apply 400 uL of Wash Solution to the column and centrifuge for 1 minute Discard the flowthrough and reassemble the spin column with its collection tube 10 Repeat Step 9 to wash column second time 11 Wash the column a third time by adding another 400 uL of Wash Solution to the column and centrifuge for 1 minute Discard the flow through and reassemble the spin column with its collection tube 12 Spin the column for 2 minutes empty at 14 000 RPM in order to thoroughly dry the resin Discard the collection tube 13 Transfer the spin column to a fresh 1 7 mL Elution tube Apply 100 uL of Elution Buffer to the column and centrifuge for 2 minutes at 2 0
14. w should it be interpreted if only the PCR control and the Isolation control showed amplification in a sample e The sample tested can be considered negative 9 What if I forgot to do a dry spin after my third 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 10 What if forgot to add the Isolation Control soC during the isolation e It is recommended that the isolation is repeated 11 What if forgot to run the Control PCR for the sample and only ran the Detection PCR and obtained a positive result e The result can be considered positive However any negative result must be verified by running the associated control PCR to ensure that it is a true negative and not a false negative due to problems with the RNA isolation or the PCR reactions Related Products Product Dirofilaria immitis PCR Detection Kit 44500 Toxoplasma gondii PCR Detection Kit 44700 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 Norgen s Leptospira interrogans PCR Detection Kit or NORGEN products in general please do not hesitate to cont
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