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Pneumocystis

1. Data The kit was initially validated using the Cepheid SmartCycler Certain of the assay performance claims were re validated on the Mx3005P platform using Optical PCR tubes and caps Agilent Technologies Cat 401428 amp 401425 and are reported below 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 50 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 The following Performance Claims were established using the Cepheid SmartCycler 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 Coryn
2. Excel Only those wells dyes which have been highlighted will be exported so ensure that all relevant required wells dyes are selected 3 6 Open the saved csv file with Excel or similar spreadsheet software 3 7 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 030 163 Version 1 2 24FEB12 English 12 For in vitro Diagnostic Use MycAssay Pneumocystis Respiratory Samples Stratagene Mx 3000 series Check the Negative Control Is the PNE Ct 39 0 or recorded as No Ct Run is contaminated Check the Negative Control ACTION Repeat the run Is the IAC Ct 29 3 33 3 Run failure ACTION Repeat the run Run failure Check the Positive Control ACTION Repeat the run Is the PNE Ct 20 0 25 0 Positive for Pneumocystis DNA Check the Patient Sample Is the PNE Ct lt 39 0 F Check the Patient Sample Negative for Pneumocystis DNA Is the IAC Ct 29 3 33 3 IAC failure ACTION Repeat sample If the same result again suspect inhibitor present in sample English 13 030 163 Version 1 2 24FEB12 MycAssay Pneumocystis Stratagene Mx3000 series For in vitro Diagnostic Use Respiratory Samples Sample PNE IAC Interpretation Further Action MycAssay Ct MycAssay Ct Negative 39 0 or Within 29 3 Negative Control Patient results Control Undetected 33 3 acceptable ar
3. Pneumocystis pneumonia Emerg Infect Dis 10 1713 20 Miller RF Allen E Copas A Singer M Edwards SG Improved survival for HIV infected patients with severe Pneumocystis jirovecii pneumonia is independent of highly active antiretroviral therapy Thorax 2006 61 716 21 English 1 030 163 Version 1 2 24FEB12 MycAssay Pneumocystis For in vitro Diagnostic Use Stratagene Mx3000 series Respiratory Samples AIDS with PCP PCP also occurs in other immunocompromised patients including recipients of solid organ transplants hypogammaglobulinaemia and chronic leukaemia Currently the diagnosis of PCP relies on microscopic methods as P jirovecii cannot be cultured in routine microbiology laboratories Bronchoalveolar lavage BAL is the preferred means of sample collection Common methods for diagnosis include immunofluorescence IF or direct fluorescence and histological staining of samples MycAssay Pneumocystis is a molecular diagnostic kit for the detection of P jirovecii based on Molecular Beacon PCR technology The whole test procedure including extraction of DNA from the clinical sample can be completed within 4 hours or only 2 hours if extracted DNA is already available This assay brings the direct benefit of enhanced laboratory efficiency combined with a rapid test leading to likely clinical benefits The diagnostic accuracy of the test depends to a great extent on sample quality Principles of the Assay Foll
4. from DNA extracted from patient respiratory samples Four groups are described Clinical Reporting The MycAssay Pneumocystis kit is intended as an aid to diagnosis of Pneumocystis pneumonia The results need to be taken in context of the clinical condition of the patient and other diagnostic tests results The following are recommended reports each depending on the assay result interpretation 030 163 Version 1 2 24FEB12 English 20 For in vitro Diagnostic Use MycAssay Pneumocystis Respiratory Samples Stratagene Mx 3000 series Outcome No 1 Pneumocystis jirovecii not detected Outcome No 2 Pneumocystis jirovecii detected Positive result State Ct value Outcome No 3 Test failed inhibitors or other unknown substance present The lower the Ct value the higher the probability of disease Ct values close to the cut off of 39 0 are more likely to represent colonisation than infection but some patients may have disease with very little P jirovecii present representing a poor specimen prior treatment or the nature of fungal load in that particular patient Limitations of Procedure The principal limitation of this procedure relates to the quality of the primary sample If the sample is very small or not collected from the affected area of lung the test will be less sensitive and may be falsely negative BAL samples should be centrifuged prior to DNA extraction from the pellet Data also demo
5. v Microscopy Diagnosis Microscopy positive Microscopy negative PCR positive 45 8 0 85 PPV PCR negative 2 33 0 94 NPV 0 96 0 80 Sensitivity Specificity Table 1 Diagnostic specificity and sensitivity of the MycAssay Pneumocystis kit compared to immunofluorescent microscopy Table 1 represents data obtained from patients with diagnosed HIV patients not infected with HIV and patients with undetermined HIV status Patients with Pneumocystis pneumonia have highly variable amounts of organism detectable the lower the Ct value the higher the likelinood of disease Patients with HIV and Pneumocystis pneumonia tend to have higher numbers of organisms detectable than patients who are not infected with the virus but the overlap is considerable The scatter plot in Figure 1 below demonstrates this overlap For completion as the data set in Table 1 included patients whose HIV status was unknown the scatter plot for this group is included in Figure 1 column 3 English 19 030 163 Version 1 2 24FEB12 MycAssay Pneumocystis For in vitro Diagnostic Use Stratagene Mx3000 series Respiratory Samples 39 oe De a R 3 37 s e 35 e e 33 e 3 31 e e e 8 e E 29 gt 27 8 i 23 21 4 19 e 17 o 1 2 3 4 Category 1 HIV Microscopy 2 HIV Microscopy 3 HIV Unknown Microscopy 4 All Microscopy Figure 1 Scatter plot of Ct values obtained
6. 10 seconds using a mini centrifuge with 0 2 mL PCR tube adapter Visually check that there are no bubbles present in the reaction mixtures Proceed to Section 2 promptly MycAssay Pneumocystis reactions are stable on the bench for up to 60 minutes Following the PCR set up ensure the work area is thoroughly cleaned using DNA decontaminating reagents 030 163 Version 1 2 24FEB12 English 8 For in vitro Diagnostic Use MycAssay Pneumocystis Respiratory Samples Stratagene Mx 3000 series 2 2 1 2 2 2 3 2 4 Performing the run Open up the MxPro software version 4 10 Insert the MycAssay Pneumocystis Myconostica Protocol CD ROM In the New Options menu select the first option Real Time Quantitative PCR Multiple Standards and click OK as shown New Options Select experiment project type Real time Quantitative PCR Multiple Standards Comparative Quantitation Calibrator C SYBR Green with Dissociation Curve C EvaGreen with Dissociation Curve Allele Discrimination SNP s Real Time C Molecular Beacon Melting Curve Plate read C Quantitative Plate Read C Plate Read Allele Discrimination Project type C Multiple Experiment Analysis 7 i I Don t show this again In the Plate Setup tab click on the Import button on the right Select the MycAssay PNE Myconostica Protocol CD ROM from the drop down list in Look In and then import the file named MycAssay Pneumocystis v3_1 mxp
7. Click Finish Once this is completed the Plate Setup should look like this example English 9 030 163 Version 1 2 24FEB12 MycAssay Pneumocystis For in vitro Diagnostic Use Stratagene Mx3000 series Respiratory Samples m Fie seng cannes i j j j ja e fe E ja je i je je fe i fasonnrae td Asean thon wet teas shorn 2 5 It is recommended that in those wells which are empty that the Well Type be set to lt blank gt from the Well Type drop down list to prevent refraction of light from the plastic interfering with the signals from those wells which do contain reactions 2 6 Repeat the import process in the Thermal Profile Setup tab to import the PCR program for this assay set the Thermal Profile Design to Custom and then Import from the same file as described in 2 4 Once this is completed the Thermal Profile Setup should look like this example 030 163 Version 1 2 24FEB12 English 10 For in vitro Diagnostic Use MycAssay Pneumocystis Respiratory Samples Stratagene Mx 3000 series 2 7 2 8 2 9 Piate Setup Thermal Profile Setup Olas Thermal Profile Estimated Run Tome p Thams Piae Dewan Pianu vah Rano Data cotacion mate ty daoorg Alportis Endporte Selection Egi Propeting Delete Tromsi potle comments Ful Scrmen Prt In the Plate Setup tab name the wells appropriately Right click on
8. In vitro Diagnostic Use MycAssay Pneumocystis Stratagene Mx3000 series my con OS I ca Respiratory Samples MycAssay Pneumocystis Stratagene Mx 3000 series Respiratory Samples REF 080 035 Intended Use MycAssay Pneumocystis is indicated for use by qualified laboratory professionals for the qualitative detection of Pneumocystis jirovecii genomic DNA extracted from respiratory specimens from the lower respiratory tract e g bronchial samples as an aid to diagnosis in adult patients suspected of having P jirovecii pneumonia MycAssay Pneumocystis has been validated for use with the Stratagene Mx3000 series instruments either Mx3000P or Mx3005P in combination with MxPro software version 4 10 Summary and Explanation Pneumocystis jirovecii formerly carinii pneumonia PCP is a common opportunistic pneumonia in immunocompromised patients especially those with advanced HIV infection and AIDS It is typically community acquired and sub acute in presentation leading to progressive respiratory failure and death if untreated Prophylaxis with trimethoprim sulphamethoxazole Bactrim or Septrin is routinely given to many at risk patients a practice which has substantially reduced the incidence of PCP but breakthrough occurs and those who do not know they are HIV positive may present with 1 Morris A Lundgren JD Masur H Walzer PD Hanson DL Frederick T Huang L Beard CB Kaplan JE 2004 Current epidemiology of
9. a highlighted well or group of wells if replicates and select Well Information from the list of options Type in the name of the sample used in that well in the Name section When all the wells are named appropriately save the run giving it an appropriate file name that includes the date and the operator s initials and then start the run by selecting the Run button on the top right of the screen in the Instrument tab followed by clicking on the Start button in the bottom right corner Remember to allow the instrument to cool for at least 1 hour before starting at step 1 1 again English 11 030 163 Version 1 2 24FEB12 MycAssay Pneumocystis For in vitro Diagnostic Use Stratagene Mx3000 series Respiratory Samples 3 Data Analysis and Interpretation 3 1 Once the run has finished the results can be viewed by selecting the Analysis button on the top right of the screen followed by the Results tab 3 2 With the Amplification Plots analysis area selected set the thresholds for each channel as follows and lock by clicking on the padlock icon PNE 500 IAC 100 3 3 The Adaptive Baseline should also be selected this is usually the default setting for this software 3 4 Save the changes Each channel can be viewed separately by clicking the boxes in the Assays Shown section in the bottom left of the screen on or off 3 5 Data can be exported for further manipulation in to Excel by File gt Export Text Report gt Export Text Report to
10. carinii pneumonia JAMA 286 2450 60 4 Huang L Morris A Limper AH Beck JM ATS Pneumocystis Workshop Participants An Official ATS Workshop Summary Recent advances and future directions in pneumocystis pneumonia PCP Proc Am Thorac Soc 2006 3 655 64 Tyagi S Kramer FR 1996 Molecular beacons Probes that fluoresce upon hybridization Nature Biotechnology 14 303 308 030 163 Version 1 2 24FEB12 English 2 For in vitro Diagnostic Use MycAssay Pneumocystis Respiratory Samples Stratagene Mx 3000 series 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 Stratagene Real Time PCR System simultaneously monitors the fluorescence emitted by each beacon Precautions The kit is for in vitro diagnostic use 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 Stratagene Mx3000 series with MxPro software version 4 10 During validation studies we have noted the following specific to this instrument o If the instrument has been used immediately before please allow the lamp to cool for at least 1 hour before setting up a MycAssay Pneumocystis run otherwi
11. dapter Plate REF 080 015 available from Myconostica 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 English 5 030 163 Version 1 2 24FEB12 MycAssay Pneumocystis For in vitro Diagnostic Use Stratagene Mx3000 series Respiratory Samples 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 Accurately note the positions of samples when multiple samples are being processed Please allow the lamp to cool for at least 1 hour after a run before setting u
12. e valid Negative 39 0 or lt 29 3 or gt 33 3 Failure in Repeat entire Control Undetected Negative Control run Negative lt 39 0 Within 29 3 Contamination Repeat entire Control 33 3 run Positive Within 20 0 N A Positive Control Patient results Control 25 0 acceptable are valid Positive lt 20 0 or gt 25 0 N A Failure in Repeat entire Control Positive Control run Patient 39 0 or Within 29 3 Negative for Report result Undetected 33 3 Pneumocystis Outcome 1 Patient lt 39 0 N A Positive for Report result Pneumocystis Outcome 2 Patient 39 0 or lt 29 3 or gt 33 3 IAC failure in Repeat sample Undetected sample Outcome 3 See Clinical Reporting Outcome 1 2 or 3 030 163 Version 1 2 24FEB12 English 14 For in vitro Diagnostic Use MycAssay Pneumocystis Respiratory Samples Stratagene Mx 3000 series Troubleshooting The Negative Control has generated a positive signal in the FAM channel Contamination occurred during the set up Results from the entire run cannot be relied upon as accurate gt 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 gt Make sure that the work area and instruments are properly decontaminated before and after use The Negative Control was incorrectly positioned in the instrument gt Take care that all the reactions are correctly ann
13. ebacterium 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 Human genomic DNA does not report a positive result with this assay 030 163 Version 1 2 24FEB12 English 18 For in vitro Diagnostic Use MycAssay Pneumocystis Respiratory Samples Stratagene Mx 3000 series Interfering Substances contraindications for use The following compounds were tested at clinically relevant concentrations and found not to inhibit the assay acteylcysteine amphotericin beclometasone dipropionate budesonide colistimethate sodium fluticasone propionate formoterol fumarate dehydrate ipratropium bromide lidocaine mannitol salbutamol sulphate salmerterol septrin trimethoprim sulphamethoxazole sodium chloride sodium cromoglicate terbutaline and tobramycin Performance Evaluation The clinical cut off at a Ct of 39 0 was established following analysis of a set of clinical samples sourced from different patient populations Clinical samples collected by bronchoaleveolar lavage BAL that had been obtained at 2 hospitals extracted using the MycXtra kit and stored were used to evaluate the performance of the MycAssay Pneumocystis kit Comparisons were made of the PCR results to immunofluorescent microscopy PCR
14. ertificate of Analysis 030 163 Version 1 2 24FEB12 English 4 For in vitro Diagnostic Use MycAssay Pneumocystis Respiratory Samples Stratagene Mx 3000 series 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 Equipment Materials required and not provided Stratagene Mx3000 series Real Time PCR System including user manual attached computer and MxPro software version 4 10 Optical PCR strip tubes Agilent Technologies Cat no 401428 Optical PCR strip caps Agilent Technologies Cat no 401425 Micro centrifuge with 0 2 mL PCR tube adapter Vortex mixer Support rack for PCR tubes Micropipettes 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 Specimen The specimen for the MycAssay Pneumocystis assay is total DNA extracted from clinical BAL samples The following DNA isolation kit and equipment supplied by Myconostica Ltd is recommended for this purpose and was used during validation MycXtra Fungal DNA Extraction kit REF 080 005 available from Myconostica Vortex Genie 2 Scientific Industries Inc New York USA Vortex A
15. gal DNA Extraction kit 4 5 There are no results for any channel with any samples or controls 030 163 Version 1 2 24FEB12 English 16 For in vitro Diagnostic Use MycAssay Pneumocystis Respiratory Samples Stratagene Mx 3000 series 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 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 productsupport lab21 com English 17 030 163 Version 1 2 24FEB12 MycAssay Pneumocystis For in vitro Diagnostic Use Stratagene Mx3000 series Respiratory Samples Performance Characteristics and Limitations Mx3000 series Analytical Performance
16. ive Control is negative out of range 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 setup 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 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 all the reactions are correctly annotated within the software and that the tube strips are placed into the machine in the correct orientation Non recommended tubes or plates were used Thresholds are only valid when using the recommended Agilent Technologies PCR strip tubes and caps Cat 401428 and 401425 4 4 Patient sample s gives IAC failure gt It is likely that the extracted test sample s contain PCR inhibitors We recommend that DNA is extracted using the MycXtra Fun
17. nstrated that a reduction in the volume of supernatant used in the extraction process achieved by the centrifugation step decreases the proportion of inhibitors entering the system Clinical Performance Evaluation has not been confirmed using the MX3005P instrument While the MycXtra Fungal DNA extraction procedure is designed to remove PCR inhibitors not all drugs or patient populations have been evaluated The procedure has not been fully assessed with sputa nor has it been assessed with induced saline samples or on samples from children False positive results may arise from external contamination of the original sample or test Such contamination could arise from P jirovecii contaminated air poor experimental technique with respect to the positive control or external especially pipette contamination with P jirovecii DNA English 21 030 163 Version 1 2 24FEB12 MycAssay Pneumocystis For in vitro Diagnostic Use Stratagene Mx3000 series Respiratory Samples As a true positive result may be obtained from patients who are transiently or persistently colonized by P jirovecii clinical judgment is required in interpretation of the test result LICENSING TopTaq Hot Start provided by QIAGEN QIAGEN is a registered trade mark of Qiagen GmbH Hilden Germany SmartCycler is a registered Trademark of Cepheid 904 Caribbean Drive Sunnyvale CA 94089 USA This product is sold under license from the Public Heal
18. orrectly 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 This kit was validated using 0 2ml 8 strip optical PCR tubes and caps Agilent Technologies Cat 401428 amp 401425 Use of other sources types of plastic could invalidate the thresholds and therefore the claims made in the IFU It is recommended that should an alternative source be used that local validation should be conducted with the positive and negative control reactions 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 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 C
19. otated within the software and that the tube strips are placed into the machine in the correct orientation Non recommended tubes or plates were used gt Thresholds are only valid when using the recommended Agilent Technologies PCR strip tubes and caps Cat 401428 and 401425 4 2 The Negative Control IAC Ct value is not within the acceptable range The PCR has been inhibited gt 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 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 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 gt 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 English 15 030 163 Version 1 2 24FEB12 MycAssay Pneumocystis For in vitro Diagnostic Use Stratagene Mx3000 series Respiratory Samples gt Thresholds are only valid when using the recommended Agilent Technologies PCR strip tubes and caps Cat 401428 and 401425 4 3 The Posit
20. owing 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 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 3 kovacs JA Gill VJ Meshnick S Masur H 2001 New insights into transmission diagnosis and drug treatment of Pneumocystis
21. p MycAssay Pneumocystis to minimise false negative results Following this cooling period the lamp will require a 20 minute warm up period as noted in 1 1 below Procedure for Use 1 Real Time PCR Set Up 1 1 To begin switch on the Stratagene Mx3000 series Real Time PCR System instrument and associated computer and launch the MxPro 4 10 software Enter usernames and passwords if required The lamp will require 20 minutes to warm up Do not start the run until the lamp is ready 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 Patient 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 Patient samples may be tested using the 5 pouches in a kit For example see Mifflin T E 2003 Setting up a PCR Laboratory In PCR Primer 2nd Ed eds Dieffenbach and Dveksler Cold Spring Harbour Laboratory Press Cold Spring Harbour NY USA 030 163 Version 1 2 24FEB12 English 6 For in vitro Diagno
22. se there can be a loss in sensitivity resulting in false negatives o There is less precision at low concentrations at and around the clinical cut off relative to other instruments we have tested 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 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 English 3 030 163 Version 1 2 24FEB12 MycAssay Pneumocystis For in vitro Diagnostic Use Stratagene Mx3000 series Respiratory Samples Low concentrations of DNA can be unstable if not stored c
23. stic Use MycAssay Pneumocystis Respiratory Samples Stratagene Mx 3000 series 1 4 1 5 1 6 1 7 1 8 1 9 Calculate the number of reactions required referring to the table below Number of Pouches Maximum number of Patient samples 6 Remove the appropriate number of pouches from the freezer Do not use any pouch that is no longer sealed If the Patient 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 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 Patient 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 Always set up the negative control first followed by the test samples The positive control should always be set up last English 7 030 163 Version 1 2 24FEB12 MycAssay Pneumocystis For in vitro Diagnostic Use S
24. th Research Institute Newark New Jersey USA and may be used under PHRI patent rights only for human in vitro diagnostics Mx3000P and Mx3005P are registered trademarks of Stratagene MxPro is a trademark of Stratagene Lab21 184 Cambridge Science Park Cambridge CB4 OGA United Kingdom Telephone 44 0 1638 552 882 Facsimile 44 0 1638 552 375 Email productsupport lab21 com 030 163 Version 1 2 24FEB12 English 22
25. tratagene Mx3000 series Respiratory Samples 1 10 1 13 Reagent and DNA volumes are shown in the table below Reaction Reagent Negative Patient 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 10W Patient Sample OL Tube 4 Black cap nn 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 reaction tubes before opening Tube 4 can reduce the risk of cross contamination Make sure all reaction tube caps are firmly closed Make a note of the positions of each sample in the strip tubes Label the first tube of each strip if more than one strip tubes are used Spin down the reaction tubes for

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