42720 - Protocol (48 preps)
Contents
1. West Nile Virus HIV F Linear Range e The linear range analytical measurement of Norgen s Cryptococcus neoformans PCR Kit was determined by analyzing a dilution series of a C neoformans quantification standard ranging from 1 x 10 copies ul to 1 x 10 copies ul e Each dilution has been tested in replicates n 4 using Norgen s Cryptococcus neoformans PCR Kit on 1X TAE 1 7 Agarose gel e The linear range of Norgen s Cryptococcus neoformans PCR Kit has been determined to cover concentrations from 1 x 10 copies ul to at least 1 x 10 copies ul of isolated DNA G Technical Support 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 Technical support can also be obtained from our website www norgenbiotek com or through email at techsupport norgenbiotek com Product Use Restriction Good laboratory practice is essential for the proper performance of this kit Ensure that the purity of the kit and reactions is maintained at all times and closely monitor all reagents for contamination Do not use any reagents that appear to be contaminated Ensure that appropriate specimen collection transport storage and processing techniques are followed for optimal performance of this test The presence of PCR inhibitors may cause false negative or invalid results Potential mutations within the target regions of the Cryptococcus neoformans ge2. ed thawing and freezing gt 2 x of the Master Mix and Positive Control should be avoided as this may affect the performance of the assay If the reagents are to be used only intermittently they should be frozen in aliquots Customer Supplied Reagents and Equipment Appropriate End point PCR Instrument DNA Purification Kit o The kit is compatible with all DNA purification kits that yield high quality inhibitor free DNA o Recommended Purification Kit Norgen Biotek s purification kits for DNA isolation including Blood Genomic DNA Isolation Mini Kit Cat 46300 Disposable powder free gloves Benchtop microcentrifuge Micropipettors Sterile pipette tips with filters PCR tubes Vortex mixer Agarose gel electrophoresis apparatus UV transilluminator with suitable gel documentation system Quality Control In accordance with Norgen s ISO 9001 and ISO 13485 certified Quality Management System each lot of Norgen s Cryptococcus neoformans PCR Kit is tested against predetermined specifications to ensure consistent product quality Warnings and Precautions Follow universal precautions All specimens should be considered as potentially infectious and handled accordingly Ensure that a suitable lab coat disposable gloves and protective goggles are worn when handling specimens and kit reagents Use sterile pipette tips with filters Use proper pipetting techniques and maintain the same pipetting pattern throughout the procedure to ens
3. ific amplification of a 278 bp region of the Cryptococcus neoformans genome In addition the kit contains a positive and a negative control to confirm the integrity of the kit reagents The detection of Cryptococcus neoformans specific DNA is based on end point PCR technology utilizing polymerase chain reaction PCR for the amplification of specific Cryptococcus neoformans DNA sequences For analysis of the PCR data the PCR reaction is loaded on an agarose DNA gel along with the provided DNA ladder for qualitative analysis Norgen s Cryptococcus neoformans PCR Kit was developed and validated to be used with the following PCR instruments e Qiagen Rotor Gene Q e BioRad iCycler Kit Components Component Product 42720 48 preps 2X PCR Master Mix 2 x 350 uL C neoformans Primer Set Mix 150 uL C neoformans Positive Control 100 uL Nuclease Free Water 1 25 mL DNA Ladder 200 uL Product Insert 1 Storage Conditions and Product Stability e The Cryptococcus neoformans PCR Kit is shipped on dry ice The components of the kit should be frozen upon arrival If one or more of the components is not frozen when the kit is received or if any of the components have been compromised during shipment please contact Norgen Biotek for assistance e All kit components should be stored at 20 C upon arrival All kit components should be stored at 20 C for up to 1 year without showing any reduction in performance Repeat
4. nome covered by the primers in this kit may result in failure to detect the presence of the pathogen The respective user is liable for any and all damages resulting from application of Norgen s Cryptococcus neoformans PCR Kit for use deviating from the intended use as specified in the user manual All products sold by Norgen Biotek are subjected to extensive quality control procedures and are warranted to perform as described when used correctly Any problems should be reported immediately The kit contents are for laboratory use only and they must be stored in the laboratory and must not be used for purposes other than intended The kit contents are unfit for consumption Norgen Biotek Corp 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 2014 Norgen Biotek Corp P142720 1
5. ow the 278 bp band corresponding to the Cryptococcus neoformans target amplicon o Positive Control shows the 278 bp band o Negative Control shows no bands e Negative Sample A sample is determined to be negative only when o Sample lanes contain no bands o Positive Control shows the 278 bp band o Negative Control shows no bands Invalid Test Run e Atest run is invalid if o The run has not been completed o Positive Control does not show the 278 bp band o Negative Control shows any amplification M 1 2000 1500 1000 750 500 300 150 Figure 1 A representative 1X TAE 2 agarose gel showing the amplification of Cryptococcus neoformans The size of the Cryptococcus neoformans target amplicon corresponds to the 278 bp band represented by the provided DNA Marker M No amplification of the target is observed in with the Negative Control 2 Posc Nesc M E Specificity The specificity of Norgen s Cryptococcus neoformans PCR Kit is first and foremost ensured by the selection of the C neoformans 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 pathogens Pneumocystis jirovecii Neisseria gonorrhoea Chlamydia trachomatis Norovirus
6. p to 64 PCR reactions 48 sample PCR 8 positive control PCR and 8 no template control PCR e For every PCR run one reaction containing Cryptococcus neoformans 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 of sample DNA than recommended may affect the sensitivity of the Cryptococcus neoformans Limit of Detection e To avoid any contamination while preparing the PCR assay follow the order outlined in Tables 1 2 and 3 below to prepare the Negative Control Detection Assay and Positive Control 1 Prepare the PCR Negative Control Table 1 2 Prepare the PCR Cryptococcus neoformans Assay Table 2 3 Prepare the PCR Positive Control Table 3 e To further avoid contamination add the components to the PCR tubes in the order shown in the tables below ie 1 Nuclease free water 2 Master Mix 3 Primer Set and 4 the Sample DNA or Positive Control 1 For each PCR set prepare one no template control PCR as shown in Table 1 below Table 1 PCR Negative Control Preparation PCR Components Volume Per PCR Reaction Nuclease Free Water 8 uL Total Volume 20 uL 2 Prepare the PCR reaction for sample detection as shown in Table 2 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
7. template Adjust the final volume of the PCR reaction to 20 uL using the Nuclease Free Water provided Table 2 PCR Cryptococcus neoformans Assay Preparation PCR Components Volume Per PCR Reaction Nuclease Free Water 5 5 uL Sample DNA 2 5 uL Total Volume 20 uL 3 For each PCR set prepare one positive control PCR as shown in Table 3 below Table 3 PCR Positive Control Preparation PCR Components Volume Per PCR Reaction Nuclease Free Water 3 uL Total Volume 20 uL C Cryptococcus neoformans PCR Assay Programming 1 Program the thermocylcer according to the program shown in Table 4 below 2 Run one step PCR Table 4 Cryptococcus neoformans 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 Cryptococcus neoformans PCR Assay Interpretation 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 Ladder provided e 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 Valid Test Run e Positive Sample A sample is determined to be positive only when o Sample lanes sh
8. thy R 3430 Schmon Parkway N E N Thorold ON Canada L2V 4Y6 i Phone 905 227 8848 BIOTEK wie CORPORATION Fax 905 227 1061 Email techsupport norgenbiotek com Cryptococcus neoformans PCR Kit Product Insert Product 42720 Background Information Cryptococcus neoformans is an encapsulated yeast Infection with C neoformans is known as cryptococcosis and is the cause of the most common life threatening meningitis in patients with weakened immune systems particularly in advanced HIV AIDS C neoformans is commonly found in soil throughout the world Human infection of C neoformans occurs via inhalation of aerosolized spores From the lungs C neoformans is spread hematogenously to the Central Nervous System CNS resulting in meningoencephalitis Although the availability of antiretroviral therapy in the developed world has reduced the incidents of cryptococcosis it is still a major problem in developing countries and is one of the leading causes of death in patients with HIV AIDS In fact one of the major challenges in treating cryptococcosis is that many patients with cryptococcal CNS disease are asymptomatic in terms of cryptococcal pneumonia making it difficult for early detection Product Description Norgen s Cryptococcus neoformans PCR Kit is a research use only kit based on the use of end point PCR technology for the detection of Cryptococcus neoformans specific DNA The kit includes Master Mix and primers for the spec
9. ts that appear to be contaminated e Ensure that appropriate specimen collection transport storage and processing techniques are followed for optimal performance of this test Instructions for Use A Sample Preparation Purified DNA is the starting material for Norgen s Cryptococcus neoformans PCR Kit The quality of the DNA template will have a major impact on the performance of the kit The user must ensure that the method used for DNA purification is compatible with end point PCR technology We recommend the use of Norgen s purification kits for DNA isolation including Norgen s Blood Genomic DNA Isolation Mini Kit Cat 46300 If using a different spin column based sample preparation procedure that includes ethanol based wash buffers a column drying step consisting of centrifugation for 10 minutes at 14 000 x g 14 000 RPM using a new collection tube is highly recommended prior to the elution of the DNA This will help to prevent the carry over of any ethanol into the purified DNA as ethanol is known to be a strong inhibitor of PCR Ensure that any traces of ethanol from the sample preparation steps are eliminated prior to the elution of the DNA B PCR Assay Preparation Notes e Before use suitable amounts of all PCR components should be completely thawed at room temperature mixed by gentle vortexing or by pipetting and centrifuged briefly e Work quickly on ice e The amount of 2X PCR Master Mix provided is enough for u
10. ure optimal and reproducible values As contamination of 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 Do not use supplies and equipment across the dedicated areas of i specimen extraction ii reaction set up and iii amplification detection No cross movement should be allowed between the different areas Personal protective equipment such as laboratory coats and disposable gloves should be area specific 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 Dispose of unused kit reagents and human specimens according to local provincial or federal regulations Do not substitute or mix reagents from different kit lots or from other manufacturers Do not use components of the kit that have passed their expiration date e The presence of PCR inhibitors may cause false negative or invalid results e Potential mutations within the target regions of the Cryptococcus neoformans genome covered by the primers in this kit may result in failure to detect the presence of the pathogen e Good laboratory practice is essential for the proper performance of this kit Ensure that the purity of the kit and reactions is maintained at all times and closely monitor all reagents for contamination Do not use any reagen
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