Urine-Based Trichomonas vaginalis PCR Detection Kit
Contents
1. Gel running time will be vary depending on an electrophoresis apparatus 3 Sample results are provided below M 1 2 3 4 5 NTC Figure 1 A representative 1X TAE 1 5 agarose gel showing the amplification of Trichomonas at different concentrations Trichomonas Target The size of the Trichomonas target amplicon corresponds to 335 bp as represented by the provided DNA Marker M NTC Negative Control M 1 2 3 4 5 6 NTC 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 Trichomonas IsoC Band PCRC Band Target Band 335 bp Positive Control Negative Control For results obtained that are not covered in Table 5 above please refer to the Troubleshooting Section2. cross movement should be allowed between the different areas Personal protective equipment such as laboratory coats and disposable gloves should be area specific As contamination of patient specimens or reagents can produce erroneous results it is essential to use aseptic techniques Pipette and handle reagents carefully to avoid mixing of the samples e Use proper pipetting techniques and maintain the same pipetting pattern throughout the procedure to ensure optimal and reproducible values e Do not substitute or mix reagents from different kit lots or from other manufacturers 1 Specimen Collection and Sample Storage e 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 e 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 e Alternatively urine samples collected using any other collection and preservation systems or reagents are also compatible with this kit 2 Sample Transport e 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 e The samples should be transported foll
3. 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 Urine Based Trichomonas vaginalis PCR Detection Kit including the TRI 2x PCR Master Mix Control 2x PCR Master Mix Isolation Control and TRI Positive Control are tested against predetermined specifications to ensure consistent product quality Product Use Limitations Norgen s Urine Based Trichomonas vaginalis 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 Discl
4. it be interpreted if only the Trichomonas target was amplified in a sample e lt is recommended that the isolation is repeated 8 How 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 I forgot to add the Isolation Control soC during the isolation e lt is recommended that the isolation is repeated 11 Whatif I forgot to run the Control PCR for the sample and I 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 DNA 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 Departmentis 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 Nor
5. 3430 Schmon Parkway gt K D Thorold ON Canada L2V4Y6 i Phone 866 667 4362 e 905 227 8848 Fax 905 227 1061 BIOTEK a CORPORATION Email techsupport norgenbiotek com Urine Based Trichomonas vaginalis PCR Detection Kit Product Insert Product 52000 Pathogen Information Trichomonas vaginalis is a protozoan pathogen of the human urogenital tract A substantial proportion of infections are asymptomatic necessitating reliable testing methods Trichomoniasis is also implicated in various other genito urinary syndromes including cervicitis epididymitis and prostatitis Diagnosis of trichomoniasis based solely on clinical signs and symptoms is unreliable because the spectrum of infection is broad and other sexually transmitted pathogens can cause similar signs and symptoms Diagnosis is particularly challenging in men where infections are characterized by fewer organisms than infections in women Early rapid diagnosis of this disease is essential to treat the illness before any serious complications arise Principle of the Test Norgen s Urine Based Trichomonas vaginalis PCR Detection Kit constituents a ready to use system for the isolation and detection of Trichomonas vaginalis using end point PCR The kit first allows for the isolation of T vaginalis DNA from urine samples using spin column chromatography based on Norgen s proprietary resin The T vaginalis DNA is isolated free from inhibitors and can then be used as the templat
6. Ignore any bands that appears in between the Isolation Control Band and the PCR Control Band F Specificity e The specificity of Norgen s Urine Based Trichomonas vaginalis PCR Detection Kit is first and foremost ensured by the selection of the Trichomonas specific primers as well as the selection of stringent reaction conditions The Trichomonas specific primers were checked for possible homologies to GenBank published sequences by sequence comparison analysis and published Trichomonas strains G Linear Range e The linear range of Norgen s Urine Based Trichomonas vaginalis PCR Detection Kit was determined by analysing a dilution series of a Trichomonas quantification standards ranging from 1pg to 10 ng e Each dilution has been tested in replicates n 4 using Norgen s Urine Based Trichomonas vaginalis PCR Detection Kit on a 1X TAE 1 5 agarose gel e The linear range of Norgen s Urine Based Trichomonas vaginalis PCR Detection Kit has been determined to cover concentrations from 6 copies uL to at least 6 x 10 copies uL e Under the conditions of the Norgen s Urine Based Trichomonas DNA Isolation procedure Norgen s Trichomonas vaginalis PCR Detection Kit covers a linear range from 100 copies mL urine to at least 1 x 10 copies mL urine Frequently Asked Questions 1 How many samples should be included per PCR run e Norgen s Urine Based Trichomonas vaginalis PCR Detection Kit is designed to test 24 samples For every 6 s
7. aimers 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 wv 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 wwwnorgenbiotek com 1 Protocol A Specimen Collection Storage and Transport General Precautions e Follow universal precautions All patient specimens should be considered as potentially infectious and handled accordingly Wear personal protective equipment including gloves and lab coats when handling kit reagents Wash hands thoroughly when finished performing the test Do not smoke drink or eat in areas where kit reagents and or human specimens are being used Dispose of unused kit reagents and human specimens according to local provincial or federal regulations Do not use supplies and equipment across the dedicated areas of specimen extraction and sample preparation No
8. amples a non template control Nuclease Free Water 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 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 Trichomonas 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 Trichomonas negative 5 How should it be interpreted if the PCR control and the Trichomonas 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 Trichomonas target and the PCR control were amplified in a sample e The sample tested can be considered as Trichomonas positive 7 How should
9. e in a PCR reaction for T vaginalis detection using the provided T vaginalis Master Mix The T vaginalis Mastermix contains reagents and enzymes for the specific amplification of a 335 region of the genome In addition Norgen s T vaginalis 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 solation Control IlsoC This kit is designed to allow for the testing of 24 samples Kit Components eS O O oes OE Wash Solution 9mL E a E Nuclease Free Water 1 25 mL IsoC Isolation Control PosC Positive Control a The isolation control is a cloned PCR product The positive control is gnomic DNA of Trichomonas vaginalis Customer Supplied Reagents and Equipment Disposable powder free gloves Centrifuge with a swinging bucket rotor capable of 2000 RPM Benchtop microcentrifuge Micropipettors Sterile 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 TRI 2x PCR Master Mix Control 2x PCR Master Mix Isolation Control IsoC the TRI Positive Control PosC and the Nuclease Free Water should be kept tightly sealed and stored at 20 C
10. gen s Urine Based Trichomonas vaginalis 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 techs upport 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 P152000 1
11. lamydia PCR Detection Kit should remain at 20 C until DNA is extracted and ready for PCR amplification e It is important to work quickly during this procedure 10 11 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 Centrifuge for 5 minutes at 2 000 RPM then discard the supernatant carefully in order not to dislodge the precipitated slurry pellet 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 Add 500 uL Solution B to the precipitated slurry pellet mix well by vortexing for 10 seconds Add 10 uL Isolation Control IsoC to the mixture from Step 4 Add 500 uL of 96 100 Ethanol to the mix from Step 4 mix well by vortexing for 10 seconds 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 Repeat Step 7 until the entire mixture from Step 6 has been transferred to the Mini Filter Spin Column 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 Repeat Step 9 to wash column second time Wash the column a third time by adding another 400 uL of Wash Solution to the column and centrifuge for 1
12. 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 2 000 RPM followed by 1 minute at 14 000 RPM C Trichomonas 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 TRI 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 e For each sample one PCR reaction using the TRI 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 e For every PCR run one reaction containing TRI 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 Trichomonas vaginalis Limit of Detection 1 Prepare the PCR for sample detection Set 1 using TRI 2x PCR Mastermix and control detection Set 2 using Control 2x PCR Mastermix as sh
13. owing the local and national instructions for the transport of pathogen material B Isolation of DNA from Urine Notes e Do not spin down or filter the urine sample before proceeding with the isolation as this could negatively affect the isolation of Chlamydia DNA e 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 e Preheat an incubator or heating block to 60 C e Prepare a working concentration of Wash Solution by adding 21 mL of 96 100 ethanol provided by the user to the supplied bottle containing the concentrated Wash Solution This will give you a final volume of 30 mL The label on the bottle has a box that may be checked to indicate that the ethanol has been added e Prepare a 400 mg mL stock solution approximately 1 7 x10 units mL of lysozyme as per supplier s instructions e Isolation Control IsoC e An Isolation Control IsoC is supplied This allows the user to control the DNA isolation procedure For this assay add the Isolation Control IsoC as indicated during the isolation procedure e The Isolation Control IsoC must not be added to the sample material directly e Do not freeze and thaw the Isolation Control IsoC more than 2 times e The Isolation Control IsoC must be kept on ice at all times during the isolation procedure e The PCR components of the Urine Based Ch
14. own 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 TRI 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 pL 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 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 D Trichomonas PCR Assay Programming 1 Program the thermocylcer according to the program shown in Table 4 below 2 Run one step PCR Table 4 Trichomonas PCR Assay Program ron ok Cycle 3 Cycle 4 EA za Ea o E 3205 Sept Bees E Trichomonas 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 pL 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
Download Pdf Manuals
Related Search