Pneumocystis jirovecii (carinii) - bio
Contents
1. 1 Prepare Mix of PCR buffer FRT and TaqF Polymerase by adding 30 gl of TaqF Polymerase in the tube with PCR buffer FRT This mix is stable for 3 months at 2 8 C If you don t need to use the all volume in 3 months it is possible to prepare a lower quantity of Mix of PCR buffer FRT and TaqF Polymerase at the rate of 10 to 1 for example 150 pl of PCR buffer FRT and 15 pl of TaqF Polymerase 2 Prepare required quantity of reaction tubes for samples and controls and add for each tube 10 pl of PCR mix 1 and 5 pl of Mix PCR buffer FRT with TagF Polymerase 3 Add 10 pl of extracted DNA sample to appropriate tube Re centrifuge all the tubes with extracted DNA for 2 min at maximum speed 12000 16000 g and take carefully supernatant N B don t disturb the pellet sorbent inhibit reaction 4 Prepare for each panel 2 controls e add 10 pl of DNA buffer to the tube labeled Amplification Negative Control NCA e add 10 pl of Pneumocystis jirovecii carinii and Human DNA C to the tube labeled C The results are interpreted through the presence of crossing of fluorescence curve with the threshold line B globine gene IC is detected on the FAM Green channel Pneumocystis jirovecii carinii on the JOE Yellow HEX Cy3 channel AMPLIFICATION 1 Create a temperature profile on your instrument as follows Rotor type Instruments Plate or modular type Instruments Step ieee ues Ti2. and Internal Control IC Test contains an IC B globine gene which serves as an amplification control for each individually processed specimen and to identify possible reaction inhibition Sacace Pneumocystis jirovecii carinii Real TM VER 21 03 2013 MATERIALS PROVIDED Module No 1 Real Time PCR kit P2 50FRT Part N 2 Pneumocystis jirovecii carinii Real TM Real Time Amplification PCR mix 1 Pneumocystis jirovecii carinii Glob 0 6 ml PCR buffer FRT 0 3 ml TaqF Polymerase 0 03 ml Positive Control Pneumocystis jirovecii carinii and Human DNA C 0 1 ml Negative Control C 1 2 ml DNA buffer 0 5 ml Contains reagents for 55 tests Module No 2 Complete Real Time PCR test with DNA purification kit TP2 50FRT Part N 1 DNA RNA Prep Sample preparation kit Lysis Sol 15 ml Prec Sol 20 ml Washing Sol 3 25 0 ml Washing Sol 4 10 0 ml RE buffer 4x 1 2 ml Contains reagents for 50 extractions Part N 2 Pneumocystis jirovecii carinii Real TM PCR mix 1 Pneumocystis jirovecii carinii Glob 0 6 ml PCR buffer FRT 0 3 ml TaqF Polymerase 0 03 ml Positive Control Pneumocystis jirovecii carinii and Human DNA C 0 1 ml Negative Control C 1 2 ml DNA buffer 0 5 ml Contains reagents for 55 tests must be used in the isolation procedure as Negative Control of Extraction Sacace Pneumocystis jirovecii carinii Real TM VER 21 03 2013 MATERIALS REQUIRED
3. BUT NOT PROVIDED Zone 1 sample preparation e DNA extraction kit Module No 1 e Biological cabinet e Desktop microcentrifuge for eppendorf type tubes e 60 C 2 C dry heat block e Vortex mixer e Pipettors capacity 5 40 ul 40 200 ul 200 1000 ul with aerosol barrier e 1 5 ml polypropylene sterile tubes Sarstedt QSP Eppendorf e Disposable gloves powderless e Biohazard waste container Zone 2 RT and amplification e Desktop microcentrifuge for eppendorf type tubes e Vortex mixer e Disposable gloves powderless e Biohazard waste container e Refrigerator Freezer e Real Time Thermal cycler e Reaction tubes or plate e Workstation e Pipettes adjustable e Sterile pipette tips with filters e Tube racks STORAGE INSTRUCTIONS Pneumocystis jirovecii carinii Real TM must be stored 20 C DNA RNA Prep must be stored at 2 8 C The kits can be shipped at 2 8 C for 3 4 days but should be immediately stored at 20 C and 2 8 C on receipt STABILITY Pneumocystis jirovecii carinii Real TM is stable up to the expiration date indicated on the kit label The product will maintain performance through the control date printed on the label Exposure to light heat or humidity may affect the shelf life of some of the kit components and should be avoided Repeated thawing and freezing of these reagents should be avoided as this may reduce the sensitivity QUALITY CONTROL In accordance with Sacace s ISO 13
4. that the specimen preparation the amplification and the detection steps are performed correctly If the controls are out of their expected range see table Results for Controls all of the specimens and controls from that run must be processed beginning from the sample preparation step PERFORMANCE CHARACTERISTICS Sensitivity The analytical sensitivity of Pneumocystis carinii Real TM PCR kit is 200 Pneumocystis carinii DNA copies ml N The claimed analytical features of Pneumocystis carinii Real TM PCR kit are guaranteed only when additional reagent kit DNA RNA prep is used Specificity The analytical specificity of Pheumocystis carinii Real TM PCR kit is ensured by selection of specific primers and probes as well as stringent reaction conditions The primers and probes were checked for possible homologies to all sequences published in gene banks by sequence comparison analysis The clinical specificity of Pneumocystis carinii Real TM PCR kit was confirmed in laboratory clinical tests Sacace Pneumocystis jirovecii carinii Real TM VER 21 03 2013 TROUBLESHOOTING 1 Weak or absent signal of the IC Fam Green retesting of the sample is required e The PCR was inhibited Make sure that you use a recommended DNA extraction method and follow the manufacturer s instructions Re centrifuge all the tubes before pipetting the extracted DNA for 2 min at maximum speed 12000 16000 g and take carefully supernatant e The reagents
5. 485 Certified Quality Management System each lot is tested against predetermined specifications to ensure consistent product quality Sacace Pneumocystis jirovecii carinii Real TM VER 21 03 2013 WARNINGS AND PRECAUTIONS In Vitro Diagnostic Medical Device For In Vitro Diagnostic Use Only The user should always pay attention to the following A Lysis Solution contains guanidine thiocyanate Guanidine thiocyanate is harmful if inhaled or comes into contact with skin or if swallowed Contact with acid releases toxic gas Xn R 20 21 22 36 37 38 S 36 37 39 JN Component Prec Sol contains 2 propanol flammable Irritant R10 36 67 S7 16 24 25 26 Avoid contact with skin and eyes S26 In case of contact with eyes rinse immediately with plenty of water and seek medical advice Use sterile pipette tips with aerosol barriers and use new tip for every procedure Store extracted positive material samples controls and amplicons away from all other reagents and add it to the reaction mix in a separate area Thaw all components thoroughly at room temperature before starting an assay When thawed mix the components and centrifuge briefly Use disposable gloves laboratory coats and eye protection when handling specimens and reagents Thoroughly wash hands afterwards Do not eat drink smoke apply cosmetics or handle contact lenses in laboratory work areas Do not use a kit after its expiration date Dispose of all specimens a
6. iSacace BIOTECHNOLOGIES For in Vitro Diagnostic Use C Pneumocystis jirovecii carinii Real M Handbook Real Time PCR Kit for the detection of Pneumocystis jirovecii carinii in biological materials REF P2 50FRT REF TP2 50FRT Y 50 Sacace Pneumocystis jirovecii carinii Real TM VER 21 03 2013 NAME Pneumocystis jirovecii carinii Real TM INTRODUCTION Pneumocystis pneumonia PCP or pneumocystosis is a form of pneumonia caused by the yeast like fungus which had previously been erroneously classified as a protozoan Pneumocystis jirovecii carinii P jirovecii is now one of several organisms known to cause life threatening opportunistic infections in patients with advanced HIV infection worldwide An accurate diagnosis requires access to modern medical care not available worldwide Currently the frequency of documented Pneumocystis infection is increasing in Africa with Pneumocystis organisms found in up to 80 on infants with pneumonia who have HIV infection INTENDED USE The Pneumocystis jirovecii carinii Real TM is a Real Time test for the qualitative detection of Pneumocystis jirovecii carinii in biological materials PRINCIPLE OF ASSAY Kit Pneumocystis jirovecii carinii Real TM is based on two major processes DNA extracted from samples and amplification using real time amplification with fluorescent reporter dye probes specific for Pneumocystis jirovecii carinii
7. icient LOT Lot Number areas snare IVD 5 in Vitro Diagnostic VER verion se Store at NCA egg CORIROROE 4 Amplification Manufacturer c Negative control of Extraction Consult instructions for Positive Control of C dedi use Amplification T Expiration Date IC Internal Control SaCycler is a registered trademark of Sacace Biotechnologies iQ5 is a registered trademark of Bio Rad Laboratories Rotor Gene Technology is a registered trademark of Qiagen MX3005P is a registered trademark of Agilent Technologies ABIG is a registered trademark of Applied Biosystems LineGeneKO is a registered trademark of Bioer SmartCycler is a registered trademark of Cepheid Sacace Biotechnologies Srl via Scalabrini 44 22100 Como Italy Tel 390314892927 Fax 390314892926 mail info sacace com web www sacace com Sacace Pneumocystis jirovecii carinii Real TM VER 21 03 2013
8. l and mix by vortex Centrifuge all tubes at 13 000 r min for 5 min and 9 10 11 using a micropipette with a plugged aerosol barrier tip carefully remove and discard supernatant from each tube without disturbing the pellet Change tips between the tubes Add 500 ul of Wash Sol 3 into each tube Vortex vigorously to ensure pellet washing Centrifuge all tubes at 13 000 r min for 60 sec and using a micropipette with a plugged aerosol barrier tip carefully remove and discard supernatant from each tube without disturbing the pellet Change tips between the tubes Add 200 pl of Wash Sol 4 into each tube Vortex vigorously to ensure pellet washing Centrifuge all tubes at 13 000 r min for 60 sec and using a micropipette with a plugged aerosol barrier tip carefully remove and discard supernatant from each tube without disturbing the pellet Change tips between the tubes Incubate all tubes with open caps at 65 C for 5 min Resuspend the pellet in 50 pl of RE buffer elution volume can be increased up to 90 ul Incubate for 5 min at 65 C and vortex periodically Centrifuge the tubes at 13000g for 60 sec The supernatant contains RNA DNA ready for amplification If amplification is not performed the same day of extraction the processed samples can be stored at 2 8 C for at maximum period of 5 days or frozen at 20 80 C Sacace Pneumocystis jirovecii carinii Real TM VER 21 03 2013 PROTOCOL Reaction volume 25 ul
9. lid only if the control samples results comply with the following Results for controls Ct in channel Control Stage for control FAM Green JOE Yellow HEX Interpretation NCE DNA extraction 35 Neg OK NCA Amplification Neg Neg OK C Amplification lt 35 33 OK 1 The sample is considered positive if Ct values detected in the FAM Green and JOE Yellow HEX channel are less than the boundary Ct values x 38 for these channels The fluorescence curve should have a typical sigmoid shape and cross the threshold line in the region of significant fluorescence increase only once 2 The sample is considered negative if its fluorescence curve does not cross the threshold line Ct value is absent and does not have the typical shape The results of analysis are considered reliable only if the results obtained for Positive and Negative Controls of Amplification as well as for the Negative Control of Extraction are correct Sacace Pneumocystis jirovecii carinii Real TM VER 21 03 2013 QUALITY CONTROL PROCEDURE A defined quantity of Internal Control IC is introduced into each sample and control at the beginning of sample preparation procedure in order to control the extraction process of each individual sample and to identify possible reaction inhibition A negative control of extraction NCE negative amplification control NCA positive amplification control C are required for every run to verify
10. me Repeats dico Time Repeats 1 95 15 min 1 95 15 min 1 95 5s 95 5s 2 60 20 s 5 60 20 s 5 72 15s 72 15s 95 5s 95 5s 20s 30s 3 60 fluorescent signal 40 60 fluorescent 40 detection signal detection 72 15s 72 15s For example Rotor Gene 3000 6000 Q Corbett Research Qiagen For example SaCycler 96 Sacace iQ5 BioRad Mx3005P Agilent ABI amp 7300 7500 StepOne Real Time PCR Applied SmartCycler Cepheid LineGeneK Bioer Sacace Pneumocystis jirovecii carinii Real TM VER 21 03 2013 INSTRUMENT SETTINGS Rotor type instruments RotorGene 3000 6000 RotorGene Q More Settings Channel Threshold Outlier Removal Slope Correct FAM Green 0 03 10 On JOE Yellow 0 03 10 On Plate type instruments The threshold line should cross only sigmoid curves of signal accumulation of positive samples and should not cross the baseline otherwise the threshold level should be raised Set the threshold at a level where fluorescence curves are linear and do not cross curves of the negative samples DATA ANALYSIS Pneumocystis carinii DNA amplification product is detected in the JOE Yellow HEX channel Internal Control B globine gene amplification product is detected in the FAM Green channel The results are interpreted by the software of the PCR instrument by the crossing or not crossing of the fluorescence curve with the threshold line The analysis results are considered va
11. n dates printed on the box and labels of all components Do not use a kit after its expiration date SAMPLE COLLECTION STORAGE AND TRANSPORT Pneumocystis jirovecii carinii Real TM can analyze DNA extracted from Sputum bronchial or tracheal lavage must be treated with the following procedure o Collect sputum into 50 mL single use PP tubes with a screw cap o Ina biological safety cabinet homogenize samples after mixing with equal volume of 4 NaOH solution N acetyl L cysteine may be added if required in the amount of 50 70 mg per sample Mix intensely with a tube rotator for 5 20 minutes depending on the density of a sample o Centrifuge samples at 3000 rpm 2800 3000 g for 15 min and carefully discard the supernatant leaving 500 1000 ul in the tube Resuspend sediment and transfer it into a 1 5 ml tube o Centrifuge samples at 12000 rpm for 5 10 min discard the supernatant and use the same 1 5 ml sample tube for DNA isolation from sample sediment bronchial lavage centrifuge 10 mL at 7000 g min for 10 15 min Remove and discard the supernatant If the pellet isn t visible add 10 ml of liquid and repeat centrifugation remove and discard the supernatant Resuspend the pellet in 100 ul of saline water tissue 71 0 gr homogenized with mechanical homogenizer or scalpel glass sticks teflon pestles and dissolved in 1 0 ml of saline water or PBS sterile 1 volume of tissue to 1 volumes of saline solution Vortex vigorously and inc
12. nd unused reagents in accordance with local authorities regulations Specimens should be considered potentially infectious and handled in a biological cabinet in accordance with appropriate biosafety practices Clean and disinfect all sample or reagent spills using a disinfectant such as 0 596 sodium hypochlorite or other suitable disinfectant Avoid sample or reagent contact with the skin eyes and mucous membranes If skin eyes or mucous membranes come into contact rinse immediately with water and seek medical advice immediately Material Safety Data Sheets MSDS are available on request Use of this product should be limited to personnel trained in the techniques of DNA amplification The laboratory process must be one directional it should begin in the Extraction Area and then move to the Amplification and Detection Areas Do not return samples equipment and reagents to the area in which the previous step was performed A Some components of this kit contain sodium azide as a preservative Do not use metal tubing for reagent transfer Only for Module No 2 Sacace Pneumocystis jirovecii carinii Real TM VER 21 03 2013 PRODUCT USE LIMITATIONS All reagents may exclusively be used in in vitro diagnostics Use of this product should be limited to personnel trained in the techniques of DNA amplification EN375 Strict compliance with the user manual is required for optimal PCR results Attention should be paid to expiratio
13. storage conditions didn t comply with the instructions Check the storage conditions e The PCR conditions didn t comply with the instructions Check the PCR conditions and for the IC detection select the fluorescence channel reported in the protocol e Improper DNA extraction Repeat analysis starting from the DNA extraction stage Make attention during the DNA extraction procedure 2 Weak Ct gt 38 signal on the Joe Yellow Cy3 HEX channel the result is considered equivocal It is necessary to repeat the analysis twice If a positive Ct value is detected twice the sample is considered as positive 3 Joe Yellow Cy3 HEX signal with Negative Control of extraction e Contamination during DNA extraction procedure All samples results are invalid Decontaminate all surfaces and instruments with sodium hypochlorite and ethanol Use only filter tips during the extraction procedure Change tips among tubes Repeat the DNA extraction with the new set of reagents 4 Any signal with Negative PCR Control e Contamination during PCR procedure All samples results are invalid Decontaminate all surfaces and instruments with sodium hypochlorite and ethanol or special DNA decontamination reagents Pipette the Positive controls at the end Repeat the PCR preparation with the new set of reagents Sacace Pneumocystis jirovecii carinii Real TM VER 21 03 2013 KEY TO SYMBOLS USED List Number Caution Contains suff
14. ubate 30 min at room temperature Transfer the supernatant into a new 1 5 ml tube swabs insert the swab into the nuclease free 1 5 ml tube and add 0 2 mL of Transport medium Vigorously agitate swabs in medium for 15 20 sec It is recommended to process samples immediately after collection Store samples at 2 8 C for no longer than 24 hours or freeze at 20 80 C Transportation of clinical specimens must comply with country federal state and local regulations for the transport of etiologic agents DNA ISOLATION The following kit is recommended gt DNA RNA Prep Sacace REF K 2 9 Please carry out DNA extraction according to the manufacture s instruction Sacace Pneumocystis jirovecii carinii Real TM VER 21 03 2013 SPECIMEN AND REAGENT PREPARATION reagents supplied with the module no 2 1 Prepare required number of 1 5 ml disposable polypropylene micro centrifuge tubes including one tube for Negative Control of Extraction Negative Control NCE N B if the sample is sputum use the same 1 5 ml sample tube obtained from sputum preparation procedure see SAMPLE COLLECTION STORAGE AND TRANSPORT 2 Add to each tube 300 ul of Lysis Sol 3 Add 100 ul of samples to the appropriate tubes using pipette tips with aerosol barriers Prepare Controls as follows o add 100 ul of NCE to the tube labeled Cneg Vortex the tubes and incubate for 5 min at 65 C Centrifuge for 7 10 sec 6 Add 400 pl of Prec So
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