Urine-Based Neisseria gonorrhoeae PCR Detection Kit
Contents
1. For results obtained that are not covered in Table 5 above please refer to the Troubleshooting Section Ignore any bands that appear between the Isolation Control band and the PCR Control band E Specificity The specificity of Norgen s Urine Based N gonorrhoeae PCR Detection Kit is first and foremost ensured by the selection of the N gonorrhoeae specific primers as well as the selection of stringent reaction conditions The primers were checked for possible homologies in GenBank published sequences by sequence comparison analyses Furthermore the specificity of the N gonorrhoeae specific primers were tested against most of the known sexually transmitted pathogens F Linear Range e The linear range analytical measurement of Norgen s Urine Based N gonorrhoeae PCR Detection Kit was determined by analyzing a dilution series of N gonorrhoeae quantitative standard ranging from 8 46 x 10 copies ul to 1 x 10 copies ul e Each dilution has been tested in replicates n 4 using Norgen s Urine Based N gonorrhoeae PCR Detection Kit on 1X TAE 1 7 Agarose gels e The linear range of Norgen s Urine Based N gonorrhoeae PCR Detection Kit has been determined to cover concentrations from 0 2 copies l to at least 8 x 10 copies ul e Under the conditions of Norgen s Urine DNA Isolation procedure Norgen s Urine Based N gonorrhoeae PCR detection Kit covers a linear range from 200 copies mL urine to at least 82. gonorrhoeae PCR control were amplified in a sample e The sample tested can be considered as N gonorrhoeae positive 6 How should it be interpreted if only the N gonorrhoeae targets was amplified in a sample e The sample tested can be considered positive At high N gonorrhoeae load the N gonorrhoeae amplicon will be predominant and the N gonorrhoeae PCR control as well as the N gonorrhoeae Isolation control may not amplify 7 How should it be interpreted if only the N gonorrhoeae PCR control and the N gonorrhoeae Isolation control showed amplification e The sample tested can be considered negative 8 Can process a different urine volume e The reagents provided with the isolation kit are only sufficient to process 24 urine samples of 5mL each 9 What If added more or less of the specified reagents volume during DNA isolation e Adding less volume may reduce your DNA yields Adding more may not affect the DNA yields EXCEPT if more Elution Buffer was added Eluting DNA in higher volumes of Elution Buffer will result in diluting your DNA 10 What If forgot to do a dry spin after my second wash e Your DNA elution will be contaminated with the Wash Solution This may dilute the DNA yield in your elution and it may interfere with your down stream applications 11 What If I forgot to add the N gonorrhoeae Isolation control during the Isolation The isolation must be repeated Related Products Pr
3. Fax 905 227 1061 BIOTEK ad CORPORATION Email techsupport norgenbiotek com cok 3430 Schmon Parkway lt Thorold ON Canada L2V 4Y6 z Phone 866 667 4362 905 227 8848 Urine Based Neisseria gonorrheae PCR Detection Kit Product Insert Product 30900 Neisseria gonorrhoeae is a Gram negative coccus of the Neisseria genus N gonorrhoeae is usually seen in pairs infecting human cells It has a circular DNA genome of approximately 1Mbp encoding over 2000 genes N gonorrhoeae is transmitted by sexual contact and usually causes infection in cells of the mucous membrane of the male urethra or the endocervix and urethra in females During infection polysaccharides are released from the bacteria that stimulate host cell production of tumour necrosis factors that cause an inflammatory response There is no vaccine against N gonorrhoeae infection and antibiotic resistance is beginning to increase therefore treatment includes a course of antibiotics that will be effective against resistant strains Complications in males caused by the infection can result in prostatitis or orchitis if the bacteria spread In females invasion of the fallopian tubes or ovaries can result in salpingitis or ovaritis respectively with any of these infections possibly resulting in sterility Principle of the Test Norgen s Urine Based Neisseria gonorrhoeae PCR Detection Kit constituents a ready to use system for the isolation and detection of N gonorrhoeae usi
4. 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 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 each 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 N gonorrhoeae PCR Assay Programming 1 Program the thermocylcer according to the program shown in Table 4 below 2 Run one step PCR Table 4 N gonorrhoeae Assay Program PCR Cycle Step Temperature Duration Cycle 1 Step 1 95 C 3 min Step 1 94 C 15 sec Cycle 2 40x 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 0 D N gonorrhoeae PCR Assay Interpretation 2000 1500 1000 750 500 300 150 For the analysis of the PCR data the entire 20 uL PCR reaction should be loaded on a 1X TAE 2 Agarose DNA gel along with 10 uL of Norgen s DNA Marker provided The PCR products should be resolved on the 1X TAE 2 Agarose gel at 150V for 30 minutes Gel running time will vary depending on an electrophoresis apparatus 2000 1500 1000 750 500 300 150 Figure 1 A representativ
5. an incubator or heating block to 60 C 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 Prepare a 400 mg mL stock solution approximately 1 7 x10 units mL of lysozyme as per supplier s instructions 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 lsoC 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 lsoC must be kept on ice at all times during the isolation procedure e The PCR components of the Urine Based N gonorrhoeae 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 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 previo
6. e 1X TAE 1 7 agarose gel showing the amplification of N gonorrhoeae at different concentrations Target The size of the N gonorrhoeae target amplicon corresponds to the 260bp band represented by the provided DNA Marker M Lanes A L represents samples spiked with different N gonorrhoeae concentrations Isolation Control 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 NTC Negative Control Table 5 Interpretation of PCR Assay Results Input Type Target Reaction Conirol Reaction Interpretation N gonorrhoeae IsoC Band PCRC Band Target Band 260 499bp 150 bp bp Positive Control X xX X Valid Negative i Control X Valid Sample X X X Positive Sample X Negative Sample X Negative Sample X Re Test Sample Re Test Sample X X Positive Sample X X Positive Sample X Re Test
7. ents should be completely thawed at room temperature vortexed and centrifuged briefly e The amount of N gonorrhoeae 2X Detection 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 e For each sample one PCR reaction using the N gonorrhoeae 2X Detection 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 results e For every PCR run one reaction containing N gonorrhoeae Positive Control 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 the N gonorrhoeae Limit of Detection 1 Prepare the PCR reaction for sample detection Set 1 using N gonorrhoeae 2X Detection PCR Mastermix and the PCR reaction for 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 N gonorrhoeae 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
8. illed liquid contains potentially infectious agents clean the affected area first with laboratory detergent and water and then with 1 v v sodium hypochlorite 1 Protocol A Specimen Collection Storage and Transport Precaution All samples have to be treated as potentially infectious material A Specimen Collection Storage and Transport General Precautions 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 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 Use proper pipetting techniques and maintain the same pipetting pattern throughout the procedure to ensure optimal and reproducible values Do n
9. ng end point PCR The kit first allows for the isolation of total DNA including bacterial DNA from the urine samples using spin column chromatography based on Norgen s proprietary resin The bacterial DNA is isolated free from inhibitors and can then be used as the template in a PCR reaction for N gonorrhoeae detection using the provided N gonorrhoeae Master Mix The N gonorrhoeae Master Mix contains reagents and enzymes for the specific amplification of a 260 bp region of the N gonorrhoeae s PorA gene In addition Norgen s Urine Based N gonorrhoeae PCR Detection Kit 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 lsoC The amplification and detection of either the Isolation Control IsoC or the PCR control PCRC does not reduce the detection limit of the analytical N gonorrhoeae PCR The kit is designed to allow for the testing of 24 samples Kit Components Component Contents 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 1 IsoC Isolateion Control PosC Positive Control The isolation control is a cloned PCR product The po
10. oduct Urine DNA Isolation Kit 18100 Urine DNA Isolation Kit for Exfoliated Cells or Bacteria 40750 Urine Exfoliated Cell RNA Purification Kit 22500 Urine Bacteria RNA Purification Kit 23400 Urine Protein Concentration Micro Kit 17400 Urine Protein Concentration Maxi Kit 21600 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 based N gonorrhoeae 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 References Morimoto M Yanai H Chiba H Matsuno K and Shukuya K 2003 Importance of midstream clean catch technique f
11. or urinalysis reconfirmed by urinary flow cytometry Clin Chim Acta 333 101 102 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 PI30900 9
12. ot substitute or mix reagents from different kit lots or from other manufacturers 1 Specimen Collection and Sample Storage 2 Sa 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 mple 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 Isolation of DNA from Urine Notes N Oo Mf A 10 11 12 13 e Do not spin down or filter the urine sample before proceeding with the isolation as this could negatively affect the isolation of N gonorrhoeae 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 Preheat
13. rdance with Norgen s ISO 9001 and ISO 13485 certified Quality Management System each lot of Norgen s N gonorrhoeae 2X PCR Master Mix Control 2X PCR Master Mix Isolation Control IsoC and N gonorrhoeae Positive Control PosC are tested against predetermined specifications to ensure consistent product quality Product Use Limitations Norgen s Urine based N gonorrhoeae 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 This kit is designed for research purposes only It is not intended for human or diagnostic use The Lysis Solution contains guanidinium salts and should be handled with care Guanidinium salts form highly reactive compounds when combined with bleach thus care must be taken to properly dispose of any of these solutions Ensu re 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 If liquid containing these buffers is spilled clean with suitable laboratory detergent and water If the sp
14. sitive control is a fragment of N gonorrhoeae cloned in a plasmid Customer Supplied Reagents and Equipment e 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 N gonorrhoeae 2X PCR Master Mix Control 2X PCR Master Mix Isolation Control IsoC and N gonorrhoeae 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 while 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 acco
15. usly 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 5 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 minute Discard the flow through and reassemble the spin column with its collection tube Spin the column for 2 minutes empty at 14 000 RPM in order to thoroughly dry the resin Discard the collection tube 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 N gonorrhoeae PCR Assay Preparation Notes e Before use suitable amounts of all PCR compon
16. x 10 copies mL urine G Frequently Asked Questions 1 How many samples should be included per PCR run e Norgen s Urine Based N gonorrhoeae PCR Detection Kit is designed to test 24 samples For every 6 samples a Negative Control and a N gonorrhoeae Positive Control PosC must be included It is preferable to pool and test 6 samples at a time If not the provided N gonorrhoeae Positive Control PosC is enough to run 3 samples at a time 2 How can interpret my results for a sample if neither the N gonorrhoeae PCR control nor the N gonorrhoeae Isolation Control amplifies e If neither the N gonorrhoeae PCR control PCRC nor the N gonorrhoeae Isolation Control lsoC 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 the problem has occurred during the setup of the PCR assay reaction 3 How should it be interpreted if only the N gonorrhoeae PCR control showed amplification but neither the N gonorrhoeae targets nor the N gonorrhoeae 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 N gonorrhoeae Isolation Control was amplified in a sample e The sample tested can be considered as N gonorrhoeae negative 5 How should it be interpreted if only the N gonorrhoeae targets and the N
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