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Pneumocystis

1. 5 Remove 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 1 6 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 5 030 148 Version 1 2 26MAR10 MycAssay Pneumocystis For Research Use Only Cepheid SmartCycler Not for use in diagnostic procedures 1 7 1 8 1 9 1 10 1 13 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 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 SmartCycler reaction tubes in their support rack s Never touch the diamond shaped reaction chamber of the reaction tube 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 R
2. Never use the reagents 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 anew 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 030 148 Version 1 2 26MAR10 English 2 For Research Use Only MycAssay Pneumocystis Not for use in diagnostic procedures Cepheid SmartCycler 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 co
3. reaction run the sample again plus the Positive and Negative controls If the reaction continues to fail an inhibiting substance may be present in the template and a Negative result cannot be relied upon 3 6 Data can be exported for analysis with a spreadsheet package using the Export button If a hardcopy of the results is also required click on Report and Print English 11 030 148 Version 1 2 26MAR10 MycAssay Pneumocystis For Research Use Only Cepheid SmartCycler 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
4. use on the block To determine how long the run will take to complete click on the Check Status tab The run name and subsequent run time will be listed SmartCycler Dx Diagnostic software version 3 0 Open up the SmartCycler Dx Diagnostic software version 3 0 and enter your username and password Insert the MycAssay Pneumocystis Myconostica Protocol CD ROM and click on the Define Assays tab Next Import the MycAssay Pneu RUO Dx 3 0 v 3 1 sca file from the CD ROM Click on the Create Run tab Enter an appropriate Run Name it is recommended that this includes the date and operators initials as a minimum or leave blank if you wish the name to be created automatically by the software Select MycAssay Pneu RUO Dx 3 0 v3 1 as the assay Enter the Lot Number and Expiration Date of the kit as printed on the kit box and each pouch The lot number will be in the form of M XXXXXXXX Enter the Number of specimens in the box and click Apply The Sample ID for each specimen will automatically be named SPEC by the software Therefore rename each site appropriately for identification purposes i e double click on SPEC to highlight it and then type in the sample ID Carefully place the reaction tubes into the designated sites in the SmartCycler block and click Start Run N B Take care when placing the reaction tubes into the designated sites as they may not be in the same order 030 148 Version 1 2 26MAR10 English 10 For Research Us
5. 7b v3 1 assay will be available for use when creating a new run Click on the Create Run tab Enter an appropriate Run Name it is recommended that this includes the date and operators initials as a minimum or leave blank if you wish the name to be created automatically by the software Select MycAssay Pneumocystis Dx1 7 v3 1 as the assay Enter the Lot Number and Expiration Date of the kit as printed on the kit box and on each pouch The lot number will be in the form of M XXXXXXXX Enter the Number of specimens in the box and click Apply The Sample ID for each specimen will automatically be named SPEC by the software English 9 030 148 Version 1 2 26MAR10 MycAssay Pneumocystis For Research Use Only Cepheid SmartCycler Not for use in diagnostic procedures 2 1 11 2 1 12 2 2 2 2 1 2 2 2 2 2 3 2 2 4 2 2 5 2 2 6 2 2 7 2 2 8 Therefore rename each site appropriately for identification purposes i e double click on SPEC to highlight it and then type in the sample ID The software will automatically include a Negative and Positive control in the Real Time PCR run Carefully place the reaction tubes into the designated sites in the SmartCycler block and click Start Run N B Take care when placing the reaction tubes into the designated sites as they may not be in the same order as your set up Make a note of the run name and click OK The run will now start and red lights will appear above each site in
6. CR Primer 2nd Ed eds Dieffenbach and Dveksler Cold Spring Harbour Laboratory Press Cold Spring Harbour NY USA 030 148 Version 1 2 26MAR10 English 4 For Research Use Only MycAssay Pneumocystis Not for use in diagnostic procedures Cepheid SmartCycler Take care to identify the SmartCycler reaction tubes appropriately when multiple samples are being processed Procedure for Use 1 Real Time PCR Set Up 1 1 To begin switch on the SmartCycler Real Time PCR System instrument and associated computer and launch the relevant software Enter usernames and passwords as required 1 2 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 1 3 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 1 4 Calculate the number of reactions required referring to the table below Number of Pouches Maximum number of test samples 6 1
7. For Research Use Only Not for use in diagnostic procedures MycAssay Pneumocystis myconos ica Cepheid SmartCycler MycAssay Pneumocystis Cepheid SmartCycler REF 080 075 Intended Use MycAssay Pneumocystis is a Real Time PCR kit for the detection of Pneumocystis jirovecii genomic DNA using the Cepheid SmartCycler Dx software versions 1 7b and 3 0 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 fluorophore when in c
8. Smart Cycler Dx User Logs Setup Archive Run s Optical Calibration Installation Qualification Complete Backup Control Trending Complete Restore System Monitoring Report Compact Database Retrieve Run s This will retrieve the selected run s into the main database Would you like to continue _caneei_ proceed 2 1 4 Select the file MycAssay Pneumocystis Dx1_7 v3_1 DXA from the CD ROM as shown below This file should be the only one recognised by the software an example is shown below Lookin E MycAssayPne Protocol CD ROM E c fat c Bee L Mycassay Pneumocystis Dx 1_7 v3_1 DXA File Name MycAssay Pneumocystis Dx 1_7 v3_1 DXA v Files of Type Smart Cycler Archive File dxa scr On the next screen highlight the filename Validation test 1 Myc Pne v3 1 and 2 1 5 click OK followed by Proceed and OK 030 148 Version 1 2 26MAR10 English 8 For Research Use Only MycAssay Pneumocystis Not for use in diagnostic procedures Cepheid SmartCycler 2 1 8 2 1 9 2 1 10 Select Run s to Retrieve Name Status User Date alidation test 1 Myc Pne v3 1 Done Unknown Jan 7 2010 02 09 PM Retrieve Run s A total of 1 run s have been selected This process takes some time to finish Would you like to continue Close the software When it is reopened the MycAssay Pneumocystis Dx1
9. arum Legionella pneumophila Moraxella catarrhalis Mycoplasma pneumoniae Neisseria meningitidis Pseudomonas aeruginosa Staphylococcus aureus Streptococcus pneumoniae S pyogenes S salivarius 030 148 Version 1 2 26MAR10 English 14 For Research Use Only MycAssay Pneumocystis Not for use in diagnostic procedures Cepheid SmartCycler 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 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 15 030 148 Version 1 2 26MAR10
10. e Only MycAssay Pneumocystis Not for use in diagnostic procedures Cepheid SmartCycler as your set up Make a note of the run name and click OK The run will now start and red lights will appear above each site in use on the block To determine how long the run will take to complete click on the Check Status tab The run name and subsequent run time will be listed 3 Data Analysis and Interpretation 3 1 The results can be viewed in Dx software by selecting the View Results tab 3 2 Click on the View Another Run button at the bottom of the page select the run you wish to view then click OK 3 3 The Sample Results tab shows the overall Assay Result and these can be interpreted using the table below Result Colour Interpretation Negative Green Negative for Pneumocystis Positive Red Positive for Pneumocystis Unresolved Yellow v3 or IAC failure in sample Repeat sample Light Grey v1 7b Invalid Light Grey Failure in Positive or Negative Control Repeat entire run Valid White Controls have performed within defined parameters Test sample results are valid 3 4 To view the Ct results for either Pneumocystis or IAC separately click on the individual tabs for each target lt PNE gt and lt IAC gt Amplification plots can also be viewed by selecting the FAM or Ch1 Optics and CY3 or Ch2 Optics channels respectively 3 5 If a sample reports an Unresolved result this is due to a failed IAC
11. eagent Negative Test Positive control sample control Tube 1 Orange cap 7 5 uL 7 5 uL 7 5 uL Tube 2 Blue cap 7 5 uL 7 5 uL Tube 3 Clear cap 10 uL Test sample 10 uL Tube 4 Black cap Total volume 25 uL 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 030 148 Version 1 2 26MAR10 English 6 For Research Use Only MycAssay Pneumocystis Not for use in diagnostic procedures Cepheid SmartCycler reaction tubes before opening Tube 4 can reduce the risk of cross contamination 1 14 Make sure all the SmartCycler reaction tube lids are firmly closed and then label each lid using a permanent marker pen e g POS for positive control NEG for negative control and an ID for the
12. g 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 It is likely that the test sample s contain PCR inhibitors Use a different DNA extraction method that removes inhibitors 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 The equipment used is not functioning optimally 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 Please refer to Section 2 and choose the correct Protocol file as specified for each software type version from the Myconostica Protocol CD ROM Only English 13 030 148 Version 1 2 26MAR10 MycAssay Pneumocystis For Research Use Only Cepheid SmartCycler Not for use in diagnostic procedures the file app
13. 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 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 4 3 The Positive Control is negative The storage conditions of the kit did not comply with the instructions in the Storage section of this IFU or the kit has expired 030 148 Version 1 2 26MAR10 English 12 For Research Use Only MycAssay Pneumocystis Not for use in diagnostic procedures Cepheid SmartCycler gt 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 11 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 the run takin
14. lose 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 1 030 148 Version 1 2 26MAR10 MycAssay Pneumocystis For Research Use Only Cepheid SmartCycler 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 Cepheid SmartCycler system with Dx diagnostic software versions 1 7b and 3 0 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
15. mplex Tube 2 lt 0 01 Primers 66 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 3 030 148 Version 1 2 26MAR10 MycAssay Pneumocystis For Research Use Only Cepheid SmartCycler Not for use in diagnostic procedures Equipment Materials required and not provided Cepheid SmartCycler Real Time PCR System including user manual attached desktop computer and Dx Diagnostic software version 1 7b or 3 0d Mini centrifuge adapted specifically for SmartCycler reaction tubes Micro centrifuge Vortex mixer SmartCycler reaction tubes Support rack for SmartCycler reaction tubes Micropipet
16. ropriate 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 35 copies This value was determined using a recombinant DNA plasmid harbouring 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 Rhizomucor pusillus Rhodotonila rubra Saccharomyces cerevisiae Scedosporium apiospermum S prolificans Sporothrix schenkii Trichosporon capitatum The following bacterial species did not report a positive result Bordetella pertussis Corynebacterium diphtheriae Escherichia coli Haemophilus influenzae Lactobacillus plant
17. tes volumes required 7 5 uL 20 uL Sterile low retention filter tips 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 In P
18. test samples Spin down the reaction tubes for 10 seconds using the specially adapted mini centrifuge Visually check that there are no bubbles present in the reaction mixtures dea 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 Before proceeding with the following section please check which version of the Dx software you have installed on your computer Open the software choose Help from the toolbar and click About For version 1 7b follow the instructions below in Section 2 1 For version 3 0 follow the instructions below in Section 2 2 Please also be aware that certain user privileges are required in the software to Retrieve Run s or Import an assay These can only be assigned by the Administrator of the instrument 2 1 SmartCycler Dx Diagnostic software version 1 7b 2 1 1 Open up the SmartCycler Dx Diagnostic software version 1 7b and enter your username and password 2 1 2 Insert the MycAssay Pneumocystis Myconostica Protocol CD ROM and click on the Define Assays tab 2 1 3 Got to Retrieve Run s via the Tools directory on the top menu bar and click Proceed English 7 030 148 Version 1 2 26MAR10 For Research Use Only MycAssay Pneumocystis Not for use in diagnostic procedures Cepheid SmartCycler

Pneumocystis

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