Candida albicans TM SC RG iQ A MX ENG ver - bio
Contents
1. Candida albicans DNA is not detected in a sample if its Ct value is absent in the FAM channel fluorescence curve does not cross the threshold line while the Ct value in the JOE channel is less than 33 The result is invalid if the Ct value of a sample in the FAM channel is absent while the Ct value in the JOE channel is either absent or greater than the specified boundary value Ct gt 33 It is necessary to repeat the PCR analysis of such samples The result of analysis is 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 Table 2 Table 2 Results for controls Control Stage for control Ct channel Fam Ct channel Joe Interpretation NCE DNA isolation NEG POS Valid result NCA Amplification NEG NEG Valid result C Amplification POS POS Valid result Sacace Candida albicans 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 that the specimen preparation the amplification and2. 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 SPECIFICATIONS Sensitivity The analytical sensitivity of Candida albicans Real TM PCR kit is specified in the table below Clinical material DNA extraction kit Analytical sensitivity GE ml Swabs DNA sorb A 5 x 10 Genome equivalents GE of the microorganism per 1 ml of a clinical sample placed in the transport medium specified Specificity The analytical specificity of Candida albicans Real TM PCR kit is ensured by selection of specific primers and probes as well as by selection of stringent reaction conditions The primers and probes were checked for possible homologies to all sequences published in gene banks by sequence comparison analysis There were no nonspecific responses during examination of human DNA as well as DNA panel of the following microorganisms Mycoplasma hominis Lactobacillus spp Escherichia coli Staphylococcus aureus Streptococcus pyogenes Streptococcus agalactiae Neisseria gonorrhoeae Ureaplasma urealyticum Ureaplasma parvum Mycoplasma genitalium Neisseria flava Neisseria subflava Neisseria sicca Neisseria mucosa Chlamydia trachomatis Trichomonas vaginalis Gardnerella vaginalis Toxoplasma gondii HSV type 1 and 2 CMV and HPV Sacace Candi
3. For In Vitro Diagnostic Use Only The user should always pay attention to the following M 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 e Use sterile pipette tips with aerosol barriers and use new tip for every procedure e Store extracted positive material samples controls and amplicons away from all other reagents and add it to the reaction mix in a separate area e Thaw all components thoroughly at room temperature before starting an assay e When thawed mix the components and centrifuge briefly e Use disposable gloves laboratory coats and eye protection when handling specimens and reagents Thoroughly wash hands afterwards e Do not eat drink smoke apply cosmetics or handle contact lenses in laboratory work areas e Do not use a kit after its expiration date e Dispose of all specimens and unused reagents in accordance with local authorities regulations e Specimens should be considered potentially infectious and handled in a biological cabinet in accordance with appropriate biosafety practices e Clean and disinfect all sample or reagent spills using a disinfectant such as 0 5 sodium hypochlorite or other suitable disinfectant e Avoid sample or reagent contact with the skin eyes and mucous membranes If skin eyes or mucous membranes come into
4. M VER 21 03 2013
5. Repeat the DNA extraction with the new set of reagents 4 Any signal with Negative Control of PCR DNA buffer e Contamination during PCR preparation procedure All samples results are invalid Decontaminate all surfaces and instruments with sodium hypochlorite and ethanol or special DNA decontamination reagents Pipette the Positive control at last Repeat the PCR preparation with the new set of reagents Sacace Candida albicans Real TM VER 21 03 2013 KEY TO SYMBOLS USED List Number LOT Lot Number For in Vitro Diagnostic Use Store at Manufacturer Consult instructions for use Expiration Date EI HE z SaCycler is a registered trademark of Sacace Biotechnologies CFX and iQ5 are registered trademarks of Bio Rad Laboratories Rotor Gene is a registered trademark of Qiagen MX3005P is a registered trademark of Agilent Technologies ABI is a registered trademark of Applied Biosystems LineGenek is a registered trademark of Bioer SmartCycler is a registered trademark of Cepheid Sacace Biotechnologies Srl VER NCA NCE C Caution Contains sufficient for lt n gt tests Version Negative Control of Amplification Negative control of Extraction Positive Control of Amplification Internal Control via Scalabrini 44 22100 Como Italy Tel 390314892927 Fax 390314892926 mail info sacace com web www sacace com Sacace Candida albicans Real T
6. arefully discard the supernatant and leave about 200 ul of solution Resuspend the sediment Use the suspension for the DNA extraction 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 Sorb A Sacace REF K 1 1 A Please carry out DNA extraction according to the manufacture s instruction Add 10 ul of Internal Control during DNA isolation procedure directly to the sample lysis mixture Note the Sacace Internal Control is the same for all urogenital infection Real Time kits Sacace Candida albicans Real TM VER 21 03 2013 SPECIMEN AND REAGENT PREPARATION reagents supplied with the module no 2 1 Lysis Solution and Washing Solution in case of their storage at 2 8 C should be warmed up to 60 65 C until disappearance of ice crystals Prepare required quantity of 1 5 ml polypropylene tubes including one tube for Negative Control of Extraction 2 Add to each tube 10 ul of Internal Control and 300 pl of Lysis Solution 3 Add 100 pl of Samples to the appropriate tube Prepare Controls as follows e add 100 pl of C Neg Control provided with the amplification kit to the tube labeled Cneg Vortex the tubes and incubat
7. contact rinse immediately with water and seek medical advice immediately e Material Safety Data Sheets MSDS are available on request e Use of this product should be limited to personnel trained in the techniques of DNA amplification e 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 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 Candida albicans 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 expiration dates printed on the box and labels of all components Do not use a kit after its expiration date SAMPLE COLLECTION STORAGE AND TRANSPORT Candida albicans Real TM can analyze DNA extracted from e cervical urethral 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 e urine sediment collect 10 20 ml of first catch urine in a sterile container Centrifuge for 30 min at 3000 x g c
8. da albicans Real TM VER 21 03 2013 TROUBLESHOOTING 1 Weak or no signal of the IC Joe Hex Cy3 channel for the Negative Control of extraction e The PCR was inhibited Make sure that you use a recommended DNA extraction method and follow to the manufacturer s instructions Re centrifuge all the tubes before pipetting of the extracted DNA for 2 min at maximum speed 12000 16000 g and take carefully supernatant Don t disturb the pellet sorbent inhibit reaction e The reagents storage conditions didn t comply with the instructions Check the storage conditions e Improper DNA extraction Repeat analysis starting from the DNA extraction stage e The PCR conditions didn t comply with the instructions Check the PCR conditions and select for the IC detection the fluorescence channel reported in the protocol e The IC was not added to the sample during the pipetting of reagents Make attention during the DNA extraction procedure 2 Weak or no signal of the Positive Control e The PCR conditions didn t comply with the instructions Check the amplification protocol and select the fluorescence channel reported in the manual 3 Fam Green 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 between tubes
9. e for 5 min at 65 C Centrifuge for 5 7 sec If the sample is not completely dissolved it is recommended to re centrifuge the tube for 5 min at a maximum speed 12000 16000 g and transfer the supernatant into a new tube for DNA extraction 6 Vortex vigorously Sorbent and add 20 ul to each tube 7 Vortex for 5 7 sec and incubate all tubes for 3 min at room temperature Repeat this step 8 Centrifuge all tubes for 30 sec at 5000g 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 9 Add 500 ul of Washing Solution to each tube Vortex very vigorously and centrifuge for 30 sec at 10000g Remove and discard supernatant from each tube 10 Repeat step 9 and incubate all tubes with open cap for 5 10 min at 65 C 11 Resuspend the pellet in 100 pl of DNA eluent Incubate for 5 min at 65 C and vortex periodically 12 Centrifuge the tubes for 1 min at 12000g 13 The supernatant contains DNA ready for amplification If amplification is not performed in 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 PROTOCOL 1 Prepare required quantity of reaction tubes for samples N and controls N 2 2 Prepare in the new sterile tube for each sample 10 N 1 ul of PCR mix 1 FRT 5 0 N 1 of PCR Buffer FRT and 0 5 N 1 of TaqF DNA Poly
10. iSacace BIOTECHNOLOGIES w IVD For in Vitro Diagnostic Use CE Candida albicans Real TM Handbook Real Time PCR kit for qualitative detection of Candida albicans REF F1 100FRT REF TF1 100FRT Y 100 Sacace Candida albicans Real TM VER 21 03 2013 NAME Candida albicans Real TM INTRODUCTION Candida albicans is a diploid fungus that grows both as yeast and filamentous cells and a causal agent of opportunistic oral and genital infections in humans Systemic fungal infections fungemias including those by C albicans have emerged as important causes of morbidity and mortality in immunocompromised patients e g AIDS cancer chemotherapy organ or bone marrow transplantation In addition hospital acquired infections by C albicans have become a cause of major health concerns INTENDED USE Kit Candida albicans Real TM is a test for the qualitative detection of Candida albicans in the urogenital swabs urine prostatic liquid and other biological materials PRINCIPLE OF ASSAY Kit Candida albicans Real TM is based on two major processes isolation of DNA from specimens and Real Time amplification Candida albicans DNA is extracted from the specimens amplified in Real Time PCR and detected using fluorescent reporter dye probes specific for Candida albicans DNA and Internal Control Internal Control IC serves as an amplification control for each individually processed specimen and to identify possible reacti
11. merase Vortex and centrifuge for 2 3 sec 3 Add to each tube 15 pl of Reaction Mix and 10 ul of extracted DNA sample to appropriate tube Mix by pipetting 4 Prepare for each panel 2 controls e add 10 pl of DNA buffer to the tube labeled Amplification Negative Control e add 10 ul of Positive Control C to the tube labeled Amplification Positive Control 5 Insert the tubes in the thermalcycler Sacace Candida albicans Real TM VER 21 03 2013 Amplification 1 Create a temperature profile on your instrument as follows Rotor type Instruments Plate or modular type Instruments Step Ee Time Repeats li EE Time Repeats 1 95 15 min 1 95 15 min 1 95 5s 95 5s 2 60 20s 5 60 20s 5 72 15s 72 15s 95 5s 95 5s 20s 30s 3 60 fluorescent 40 60 fluorescent 40 signal detection signal detection 72 15s 72 15s For example Rotor Gene 3000 6000 Q Corbett Research Qiagen For example SaCycler 96 Sacace CFX iQ5 BioRad Mx3005P Agilent ABI 7300 7500 StepOne Real Time PCR Applied Biosystems SmartCycler Cepheid LineGenek Bioer Fluorescence is detected at the 2nd step of Cycling 2 stage 60 C in FAM Green and JOE Yellow Hex Cy3 fluorescence channels Candida albicans is detected on the FAM Green channel C DNA on the JOE Yellow HEX Cy3 channel INSTRUMENT SETTINGS Rotor type instruments Calibrate Gain More Settings SETI Opti
12. misation Weal Outlier Removal Spa Gale FAM Green from 5 FI to 10 FI 0 1 5 Off JOE Yellow from 4 FI to 8 FI 0 1 5 Off 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 Boundary value of the cycle threshold Ct Channel for Ct boundary value Sample fluorophore Rotor type Plate type instruments instruments C4 FAM Green 33 36 JOE Yellow Hex Cy3 30 33 Clinical samples C JOE Yellow Hex Cy3 30 33 Sacace Candida albicans Real TM DATA ANALYSIS The fluorescent signal intensity is detected in two channels The signal from the Candida albicans DNA amplification product is detected in the FAM Green channel The signal from the Internal Control amplification product is detected in the JOE Yellow HEX Cy3 channel Interpretation of results The results are interpreted by the software of the instrument by the crossing or not crossing of the fluorescence curve with the threshold line Principle of interpretation Candida albicans DNA is detected in a sample if its Ct value is present in the FAM channel The fluorescence curve should cross the threshold line in the area of exponential fluorescence growth
13. on inhibition IC is detected in a channel other than the Candida albicans DNA Sacace Candida albicans Real TM VER 21 03 2013 MATERIALS PROVIDED Module No 1 Real Time PCR kit F1 100FRT Part N 2 Candida albicans Real TM Real Time amplification e PCR mix 1 FRT 1 2 ml e PCR Buffer FRT 2 x 0 35 ml e TaqF Polymerase 2 x 0 03 ml e Pos C 0 2 ml e Negative Control C 1 2 ml e internal Control IC 1 0 ml e DNA buffer 0 5 ml Contains reagents for 110 tests Module No 2 Complete Real Time PCR test with DNA purification kit TF1 100FRT Part N 1 DNA Sorb A sample preparation e Lysis Solution 2 x 15 ml e Sorbent 2 x 1 0 ml e Washing Solution 2 x 50 ml e DNA eluent 2 x 5 ml e Transport medium 2 x 15 ml Contains reagents for 100 tests Part N 2 Candida albicans Real TM Real Time amplification e PCR mix 1 FRT 1 2 ml e PCR Buffer FRT 2 x 0 35 ml e TaqF Polymerase 2 x 0 03 ml e Pos C 0 2 ml e Negative Control C 1 2 ml e Internal Control IC 1 0 ml e DNA buffer 0 5 ml Contains reagents for 110 tests must be used in the isolation procedure as Negative Control of Extraction add 10 ul of Internal Control during the DNA isolation directly to the sample lysis mixture see DNA Sorb A REA K 1 1 A protocol Sacace Candida albicans Real TM VER 21 03 2013 MATERIALS REQUIRED BUT NOT PROVIDED Zone 1 sample preparation e DNA ex
14. traction kit Module No 1 e Biological cabinet e Desktop microcentrifuge for eppendorf type tubes e Dry heat block e Vortex mixer e Pipettes e 1 5 ml polypropylene sterile tubes e Biohazard waste container e Refrigerator e Freezer Zone 2 Real Time amplification e Real Time Thermal cycler e Reaction tubes e Workstation e Pipettes adjustable e Sterile pipette tips with filters e Desktop centrifuge with rotor for 1 5 2 0 ml tubes e Vortex mixer e Freezer refrigerator STORAGE INSTRUCTIONS Candida albicans Real TM must be stored at 2 8 C TaqF Polymerase must be stored at 16 C DNA sorb A must be stored at 2 8 C The kits can be shipped at 2 8 C but should be stored at 2 8 C and 16 C immediately on receipt STABILITY Candida albicans 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 13485 Certified Quality Management System each lot is tested against predetermined specifications to ensure consistent product quality Sacace Candida albicans Real TM VER 21 03 2013 WARNINGS AND PRECAUTIONS In Vitro Diagnostic Medical Device
Download Pdf Manuals
Related Search