Pneumocystis - Myconostica
Contents
1. Positive Control Control Test 39 0 or Within 29 0 Negative for Pneumocystis Sample Undetermined 32 7 Test lt 39 0 N A Positive for Pneumocystis Sample Test 39 0 or lt 29 0 or gt 32 7 IAC failure in sample Sample Undetermined English 13 030 157 Version 1 0 26MAR10 MycAssay Pneumocystis For Research Use Only Applied Biosystems 7500 Not for use in diagnostic procedures 4 Troubleshooting 4 1 The Negative Control has generated a positive signal in the FAM channel gt Contamination occurred during the set up Results from the entire run cannot be relied upon as accurate Repeat the entire run taking great care when adding the templates in particular the Positive Control Tube 4 to ensure that cross contamination does not occur Make sure that the work area and instruments are properly decontaminated before and after use The Negative Control was incorrectly positioned in the instrument Take care that the reaction tubes are placed in their designated sites and are annotated correctly within the software 4 2 The Negative Control IAC Ct value is not within the acceptable range gt The PCR has been inhibited Ensure that the work area and instruments are thoroughly dry after the use of decontaminating agents prior to PCR set up The storage conditions of the kit did not comply with the instructions in the Storage section of this IFU or the kit has expired Please check corr2. or controls after their expiry date Reagents and controls should not be refrozen or reused after opening Wear protective clothing and disposable gloves while handling kit reagents Avoid microbial and deoxyribonuclease DNase contamination of reagents when removing aliquots from tubes The use of sterile DNase free low retention disposable filter tipped or positive displacement pipette tips is recommended Use a new tip for each specimen or reagent Dispose of unused reagents and waste in accordance with country federal state and local regulations To avoid contamination with Pneumocystis or IAC amplicons do not open the reaction tubes post amplification Do not eat drink or smoke in areas where specimens or kit reagents are being handled Low concentrations of DNA can be unstable if not stored correctly It is recommended that DNA extractions are stored at 80 C to preserve their integrity Multiple rounds of thawing and refreezing should also be avoided whenever possible 030 157 Version 1 0 26MAR10 English 2 For Research Use Only MycAssay Pneumocystis Not for use in diagnostic procedures Applied Biosystems 7500 Kit Contents Description The kit consists of five 3 compartment sealed foil pouches each of which can be used separately Each pouch contains sufficient reagents for 8 reactions Volume Tube 1 dNTPs 66 uL Orange Cap MgCl Buffered solution of DNA Polymerase complex Tube 2 lt 0 01 Primers 66
3. uL Blue Cap lt 0 01 Molecular Beacons lt 0 0001 Internal Amplification Control IAC The IAC is a recombinant DNA plasmid harbouring a non infective sequence unrelated to either target Pneumocystis sequence Tris HCl Buffer Tube 3 Negative Control 25 uL Clear Cap Water Tube 4 Positive Control 25 uL Black Cap lt 0 0001 Positive Control DNA The Positive Control molecule is a recombinant plasmid harbouring the Pneumocystis target sequences Tris HCl Buffer The kit also contains MycAssay Pneumocystis Myconostica Protocol CD ROM Instructions for Use Certificate of Analysis Storage The kit should be stored frozen 15 to 25 C until the expiry date indicated on the kit box label at which time it should be disposed of according to local regulations Once a pouch has been opened the contents must be used immediately not re frozen or re used English 3 030 157 Version 1 0 26MAR10 MycAssay Pneumocystis For Research Use Only Applied Biosystems 7500 Not for use in diagnostic procedures Equipment Materials required and not provided Applied BioSystems 7500 Real Time PCR System including user manual attached computer and SDS software version 1 4 MicroAmp Optical 8 tube strip Applied BioSystems part number 4316567 MicroAmp Optical 8 cap strip Applied BioSystems part number 4323032 Micro centrifuge with 0 2 mL PCR tube adapter Vortex mixer Support rack for PCR tubes Micro
4. For Research Use Only Not for use in diagnostic procedures MycAssay Pneumocystis myconos ica Applied BioSystems 7500 MycAssay Pneumocystis Applied Biosystems 7500 REF 080 075 Intended Use MycAssay Pneumocystis is a Real Time PCR kit for the detection of Pneumocystis jirovecii genomic DNA using the Applied BioSystems 7500 in combination with SDS software version 1 4 Principles of the Assay Following mixing of the reagents in the MycAssay Pneumocystis kit with a sample containing Pneumocystis target DNA sequence a portion of the Pneumocystis mitochondrial ribosomal large sub unit thermocycling will result in DNA amplification occurring The assay also contains an Internal Amplification Control IAC sequence a DNA fragment not present in Pneumocystis other fungal bacterial or human genomes to detect PCR inhibitory substances and confirm the functionality of the assay reagents The amplified DNA targets are detected with Molecular Beacons single stranded oligonucleotide hybridization probes that form a stem and loop structure The loop contains a probe sequence that is complementary to a target sequence and the stem is formed by the annealing of complementary arm sequences that are located on either side of the probe sequence A fluorophore which fluoresces when excited by light of the appropriate wavelength is covalently linked to the end of one arm and a quencher which suppresses the fluorescence of the fluoro
5. In PCR Primer 2nd Ed eds Dieffenbach and Dveksler Cold Spring Harbour Laboratory Press Cold Spring Harbour NY USA 030 157 Version 1 0 26MAR10 English 4 For Research Use Only MycAssay Pneumocystis Not for use in diagnostic procedures Applied Biosystems 7500 Accurately note the positions of samples when multiple samples are being processed Procedure for Use 1 1 1 1 2 1 3 1 4 1 5 1 6 Real Time PCR Set Up To begin switch on the AB7500 Real Time PCR System instrument and associated computer and launch the SDS v1 4 software Enter usernames and passwords if required Ensure the work area has been cleaned using DNA decontaminating reagents and allowed to dry completely avoid use during assay set up as excess cleaning solution may inhibit the PCR reactions A pouch contains one each of Tube 1 Tube 2 Tube 3 and Tube 4 There are sufficient reagents in one pouch to run 8 reactions At least one positive control and one negative control reaction must be performed per run where the reagents are from a single kit lot One pouch therefore can analyse 6 test samples If more than 6 samples need to be tested more than one pouch can be used if the pouches used are from the same kit lot A maximum of 38 test samples may be tested using the 5 pouches in a kit Calculate the number of reactions required referring to the table below Number of Pouches Maximum number of test samples 6 R
6. apitatum The following bacterial species did not report a positive result Bordetella pertussis Corynebacterium diphtheriae Escherichia coli Haemophilus influenzae Lactobacillus plantarum Legionella pneumophila Moraxella catarrhalis Mycoplasma pneumoniae Neisseria meningitidis Pseudomonas aeruginosa Staphylococcus aureus Streptococcus pneumoniae S pyogenes S salivarius 030 157 Version 1 0 26MAR10 English 16 For Research Use Only MycAssay Pneumocystis Not for use in diagnostic procedures Applied Biosystems 7500 LICENSING TopTaq Hot Start provided by QIAGEN QIAGEN is a registered trade mark of Qiagen GmbH Hilden Germany This product is sold under license from the Public Health Research Institute Newark New Jersey USA and may be used under PHRI patent rights only for human in vitro diagnostics Applied BioSystems is a registered trademark of Applera Corporation or its subsidiaries in the US and or certain other countries SmartCycler is a registered Trademark of Cepheid 904 Caribbean Drive Sunnyvale CA 94089 USA M22 4SN United Kingdom Telephone 44 0 161 998 7239 Facsimile 44 0 161 902 2496 i Myconostica Limited South Court Sharston Road Sharston Manchester Email mycotech myconostica co uk English 17 030 157 Version 1 0 26MAR10
7. cAssay Detector PNE MycAssay v Line Color Detector Color x Line Color Detector Color v Analysis Settings Analysis Settings C Auto Ct C Auto Ct Manual Ct Manual Ct Threshold 4 0e 003 Threshold 1 0e 004 C Auto Baseline C Auto Baseline Manual Baseline Manual Baseline Start cycle Start cycle 3 End cycle 15 End cycle 19 Help Help 3 4 Save the changes 030 157 Version 1 0 26MAR10 English 10 For Research Use Only MycAssay Pneumocystis Not for use in diagnostic procedures Applied Biosystems 7500 3 5 Select the wells containing samples and export the Report file File gt Export gt Results as shown below 4 7500 System SDS Software Plate1 Standard Curve DA View Tools Instrument Analysis Window Help New hN fa Chose P Save hs Savers A iu u m m Import Sample Setup Crk v v u u 3 Sample Setup View Exported Results Calbeation Data p H zl Spectra Component beatin E Cts D Dissociation gt v u o a To avoid confusion save the file with the same name as used for the run file itself Remember to save the file to an appropriate location When prompted activate Export only selected wells and click OK Open the saved csv file with Excel or similar spreadsheet software Analyse each sample starting with the controls as shown in the flowchart below details can also be found in the table shown beneath the flowchart The data is established us
8. e adapter Visually check that there are no bubbles present in the reaction mixtures 1 15 Proceed to Section 2 promptly MycAssay Pneumocystis reactions are stable on the bench for up to 60 minutes 1 16 Following the PCR set up ensure the work area is thoroughly cleaned using DNA decontaminating reagents 2 Performing the run 2 1 Open up the AB 7500 SDS software version 1 4 and enter your username and password 2 2 Insert the MycAssay Pneumocystis Myconostica Protocol CD ROM 2 3 In the Quick Startup menu select the first option Create New Document 2 4 Choose the settings as shown below Select the template MycAssay Pneumocystis SDS1_4v3_1 sdt from the CD ROM via Browse 2 5 Give the run an appropriate Plate Name An example is shown below English 7 030 157 Version 1 0 26MAR10 MycAssay Pneumocystis For Research Use Only Applied Biosystems 7500 Not for use in diagnostic procedures New Document Wizard x Define Document Select the assay container and template for the document and enter the operator name and comments Assay Standard Curve Absolute Quantitation i Container 96 Well Clear z Template MycAssay Pneumocystis SDS1_4v3_1 sdt Browse RunMode Sns y Operator your name Comments Plate Name Pneumocystis_23MAR10_NAME 2 6 Click Finish A new document will open containing the PCR parameters and detectors automatically set for this assay In the Plate v
9. e run taking care in the set up stage Such errors can be detected by seeing lower levels of liquid in this reaction compared to others The Positive Control was incorrectly positioned in the instrument Take care that the reaction tubes are placed in their designated sites 4 4 Test sample s are negative and the IAC is out of range gt gt It is likely that the test sample s contain PCR inhibitors Use a different DNA extraction method that removes inhibitors Non recommended tubes or plates were used Thresholds are only valid when using the recommended MicroAmp tubes and caps 4 5 There are no results for any channel with any samples or controls gt The storage conditions of the kit did not comply with the instructions in the Storage section of this IFU or the kit has expired Please check correct storage conditions of the kit have been followed Check the expiry date of the reagents see the kit box pouch label and repeat with an unexpired kit if necessary English 15 030 157 Version 1 0 26MAR10 MycAssay Pneumocystis For Research Use Only Applied Biosystems 7500 Not for use in diagnostic procedures The equipment used is not functioning optimally gt Please check that your Real Time PCR instrument has an up to date service history and has been fully calibrated as described in its Installation and Maintenance Guide An incorrect protocol file was used during the software set up gt Plea
10. ect storage conditions of the kit have been followed Check the expiry date of the reagents see the kit box pouch label and repeat with unexpired kit if necessary Either Tube 1 or 2 reagent was not added to the PCR reaction or double the amount of Tube 2 was added Repeat the run taking care in the set up stage Such errors can be detected by seeing higher or lower levels of liquid in one reaction tube compared to others Non recommended tubes or plates were used Thresholds are only valid when using the recommended MicroAmp tubes and caps 030 157 Version 1 0 26MAR10 English 14 For Research Use Only MycAssay Pneumocystis Not for use in diagnostic procedures Applied Biosystems 7500 4 3 The Positive Control is negative gt The storage conditions of the kit did not comply with the instructions in the Storage section of this IFU or the kit has expired Please check correct storage conditions of the kit have been followed Check the expiry date of the reagents see the kit box pouch label and repeat with an unexpired kit if necessary An error occurred during step 1 12 and the Positive Control template Tube 4 was placed in the wrong reaction tube Repeat the run taking great care during the set up stage Such errors can be detected by seeing a higher level of liquid in one reaction and a lower level in another compared to normal Either Tube 1 or 2 reagent was not added to the reaction Repeat th
11. emove the appropriate number of pouches from the freezer Do not use any pouch that is no longer sealed If the test samples were frozen after extraction also remove these from the freezer Tear open the required number of pouches and remove the tubes If more than one pouch is being used but only one set of positive and negative controls are being run it is only necessary to remove Tubes 3 and 4 from one pouch Exercise caution when handling Tube 4 This contains positive English 5 030 157 Version 1 0 26MAR10 MycAssay Pneumocystis For Research Use Only Applied Biosystems 7500 Not for use in diagnostic procedures 1 7 1 8 1 9 control DNA material and contamination could cause false positive test results Allow the tubes contents to thaw by placing on the laboratory bench for 5 10 minutes ensuring that the contents of each tube are completely thawed before proceeding Vortex to mix the tubes contents and the test samples follow by a short spin in a microcentrifuge to ensure collection of all the contents at the base of the tubes before use Place the required number of PCR tubes in the support rack Never touch the bottom of the reaction tubes with your hands Always set up the negative control first followed by the test samples The positive control should always be set up last Reagent and DNA volumes are shown in the table below Reaction Reagent Negative Positive control control Tube 1 Orange ca
12. iew of the Setup tab use Well Inspector select a well and press Ctrl 1 or right click with the mouse to name the wells according to the positions of the samples in 1 14 For example 030 157 Version 1 0 26MAR10 English 8 For Research Use Only MycAssay Pneumocystis Not for use in diagnostic procedures Applied Biosystems 7500 ae Pet At a e J 0 an SOR S at A CID 2 7 When all the wells are named appropriately save the run keeping the Plate Name as the file name 2 8 Start the run in the Instrument tab by clicking on the Start button To determine how long the run will take to complete a countdown is shown next to the Start button English 9 030 157 Version 1 0 26MAR10 MycAssay Pneumocystis For Research Use Only Applied Biosystems 7500 Not for use in diagnostic procedures 3 Data Analysis and Interpretation 3 1 Once the run has finished click on the green arrow on the top menu bar to update 3 2 Open the Amplification Plot view of the Result tab On the right hand side set the thresholds for each channel as follows The thresholds are for use with MicroAmp Optical PCR strip tubes only and may vary if different plastic consumables are used PNE MycAssay 10000 IAC MycAssay 4000 The Manual Baseline should remain at 3 15 for both detectors 3 3 Click the Analyze button to activate these changes For example Data Delta Rn vs Cycle v Data Delta Rn vs Cycle x Detector IAC My
13. ing MicroAmp Optical 8 strip PCR tubes Use of different plastic consumables may void the data English 11 030 157 Version 1 0 26MAR10 MycAssay Pneumocystis For Research Use Only Applied Biosystems 7500 Not for use in diagnostic procedures Check the Negative Control Is the PNE MycAssay Ct 39 0 or Undetermined Run is contaminated Check the Negative Control ACTION Repeat the run Is the IAC MycAssay Ct 29 0 32 7 Run failure ACTION Repeat the run Run failure Check the Positive Control ACTION Repeat the run Is the PNE MycAssay Ct 20 0 26 0 Positive for Pneumocystis DNA Check the test Sample Is the PNE MycAssay Ct lt 39 0 Check the test Sample Is the IAC MycAssay Ct 29 0 32 7 Negative for Pneumocystis DNA IAC failure ACTION Repeat sample If the same result again suspect inhibitor present in sample 030 157 Version 1 0 26MAR10 English 12 For Research Use Only Not for use in diagnostic procedures MycAssay Pneumocystis Applied Biosystems 7500 Sample PNE IAC Interpretation MycAssay Ct MycAssay Ct Negative 39 0 or Within 29 0 Negative Control acceptable Control Undetermined 32 7 Negative 39 0 or lt 29 0 or gt 32 7 Failure in Negative Control Control Undetermined Negative lt 39 0 Within 29 0 Contamination Control 32 7 Positive Within 20 0 N A Positive Control acceptable Control 26 0 Positive lt 20 0 or gt 26 0 N A Failure in
14. p 7 5 uL 7 5 uL Tube 2 Blue cap 7 5 uL Tube 3 Clear cap 10 uL Test Sample Tube 4 Black cap Total volume Add reagents in the order shown in the table above Tube 1 then Tube 2 followed by the template Negative control Test sample or Positive control Take care when taking aliquots from Tube 1 the liquid is slightly viscous and can stick on the inner ridge of the tube If this happens re spin to collect the final contents in the base of the tube before attempting to remove the final aliquots Use a new pipette tip for every liquid transfer Re cap each reagent tube after use and immediately discard it and any remaining contents into a sealable clinical waste container Unused reagents cannot be saved for later use Take extra care when pipetting Tube 4 positive control DNA to ensure it does not contaminate any other reaction tube Closing the lids on the other 030 157 Version 1 0 26MAR10 English 6 For Research Use Only MycAssay Pneumocystis Not for use in diagnostic procedures Applied Biosystems 7500 reaction tubes before opening Tube 4 can reduce the risk of cross contamination 1 14 Make sure all reaction tube caps are firmly closed Make a note of the positions of each sample in the strip tubes Label for example on the lid the first tube of each strip if more than one strip tubes are used Spin down the reaction tubes for 10 seconds using a mini centrifuge with 0 2 mL PCR tub
15. phore when in close physical proximity is covalently linked to the end of the other arm Molecular Beacons do not fluoresce when they are free in solution However when they hybridise to a nucleic acid strand containing a target sequence they undergo a conformational change that enables them to fluoresce The amount of fluorescence at any given cycle or following cycling depends on the amount of specific amplicons present at that time The SmartCycler Real Time PCR System simultaneously monitors the fluorescence emitted by each beacon English 1 030 157 Version 1 0 26MAR10 MycAssay Pneumocystis For Research Use Only Applied Biosystems 7500 Not for use in diagnostic procedures Precautions The kit is for Research Use Only It is Not for Use in Diagnostic Procedures The kit is intended for use only by laboratory professionals Procedures are required for non aerosol manipulations of specimens Standard precautions and institutional guidelines should be followed in handling all samples A Material Safety Data Sheet is available from Myconostica Ltd This test is only for use with the Applied BioSystems 7500 with SDS software version 1 4 Do not use reagents or controls if the protective pouches are open or broken upon arrival Reagents and controls are not interchangeable between kits with differing lot numbers Never pool reagents or controls from different tubes even if they are from the same lot Never use the reagents
16. pipettes volumes required 7 5 uL 20 uL Sterile low retention filtertips Disposable gloves powderless Proprietary DNA decontaminating solution Permanent marker pen DNA isolation kit see below Sample The sample for the MycAssay Pneumocystis assay is Pneumocystis genomic DNA Procedural Notes Read the entire protocol before commencing The entire MycAssay Pneumocystis process excluding DNA extraction takes approximately 2 hours dependent on the number of samples tested Setting up of the test should be performed in a PCR workstation or pre PCR laboratory If a PCR workstation is not available then the test should be set up in a dedicated area of the laboratory which is regularly cleaned with DNA decontaminating reagents However avoid using DNA decontaminating reagents during the Real Time PCR set up as they can inhibit the assay Use micropipettes for the transfer of fluids Dedicated micropipettes should be used for the set up of these reactions and they should be regularly decontaminated Low retention filter tips are recommended for use to ensure that no DNA is lost during the set up procedure Exercise caution when handling Tube 4 This contains template DNA material and contamination could result in false positive test results Wear gloves at all times All tubes must be capped following use and prior to disposal 1 For example see Mifflin T E 2003 Setting up a PCR Laboratory
17. se refer to Section 2 and choose the correct Protocol file as specified for each software type version from the Myconostica Protocol CD ROM Only the file appropriate to the software can be loaded Repeat the run using the correct protocol file If you have further questions or you experience any problems please contact Technical Support mycotech myconostica co uk Performance Characteristics and Limitations Analytical Sensitivity Using the protocol described above and a recombinant Pneumocystis DNA molecule generated at Myconostica the Limit of Detection LoD for Pneumocystis was determined to be lt 30 copies This value was determined using a recombinant DNA plasmid harboring the target sequence The Pneumocystis target sequence is mitochondrial therefore there will be numerous copies per cell but it is not known how many Analytical Selectivity Analytical selectivity was tested using DNA extracted from a variety of different fungal and non fungal species The following species did not report out a positive result Alternaria alternata Aspergillus flavus A fumigatus A niger A terreus Blastomyces capitatus Candida albicans C glabrata C parapsilosis C tropicalis Cladosporium Spp Cryptococcus neoformans Doratomyces microsporus Fusarium solani Histoplasma capsulatum Rhizomucor pusillus Rhodotonila rubra Saccharomyces cerevisiae Scedosporium apiospermum S prolificans Sporothrix schenkii Trichosporon c
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