Leishmania spp. Real TM SC RG iQ A MX Eng ver - bio
Contents
1. extraction according to the manufacture s instruction Add 10 ul of Internal Control during DNA isolation procedure directly to the sample lysis mixture SPECIMEN AND REAGENT PREPARATION 1 Prepare required number of 1 5 ml disposable polypropylene micro centrifuge tubes including one tube for Negative Control of Extraction Negative Control C 2 Add to each tube 10 pl of IC Internal Control and 300 pl of Lysis Sol 3 Add 100 pl of samples to the appropriate tubes using pipette tips with aerosol barriers Prepare Controls as follows o add 100 ul of Negative Control C to the tube labeled Cneg Vortex the tubes and incubate for 5 min at 65 C Centrifuge for 7 10 sec 6 Add 400 ul of Prec Sol 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 gl 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 plugg2. ml e TaqF Polymerase 0 03 ml e Pos Leishmania spp C 0 1 ml e Negative Control C 2 x 0 5 ml e Internal Control IC 0 6 ml e DNA buffer 0 5 ml Contains reagents for 55 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 RNA Prep REA K 2 9 protocol Sacace Leishmania spp Real TM VER 21 03 2013 MATERIALS REQUIRED 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 Dry heat block e Vortex mixer e Pipettes e Sterile pipette tips with filters e 1 5 ml polypropylene sterile tubes e Biohazard waste container e Refrigerator 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 Leishmania spp Real TM must be stored at 20 C DNA RNA Prep must be stored at 2 8 C The kit can be shipped at 2 8 C but should be stored at 2 8 C and 20 C immediately on receipt Store DNA RNA Prep kit at 2 8 C STABILITY Leishmania spp Real TM is stable up to the expiration date indicated on the kit label The product will maintain performance throu
3. samples Sacace Leishmania spp Real TM VER 21 03 2013 DATA ANALYSIS The fluorescent signal intensity is detected in two channels The signal from the Leishmania spp DNA amplification product is detected in the JOE Yellow HEX Cy3 channel The signal from the Internal Control amplification product is detected in the FAM Green channel Interpretation of results The results are interpreted through the presence of crossing of fluorescence curve with the threshold line To set threshold put the line at such level where curves of fluorescence are linear Results are accepted as relevant if positive and negative controls of amplification and extraction are passed Results for controls Control Stage for control EZ Interpretation NCE DNA isolation POS NEG Valid result NCA Amplification NEG NEG Valid result C Amplification POS POS Valid result Leishmania spp DNA is detected in a sample if its Ct value is defined in the results grid in the JOE Yellow HEX Cy3 channel Leishmania spp DNA is not detected in a sample if its Ct value is not defined in the results grid in the JOE Yellow HEX Cy3 the fluorescence curve does not cross the threshold line whereas the Ct value in the in the results grid is defined The result of analysis is invalid if the Ct value is not defined in the results grid the fluorescence curve does not cross the threshold line in the FAM Green channel I
4. CR 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 Leishmania spp Real TM VER 21 03 2013 Sacace Leishmania spp Real TM VER 21 03 2013 Sacace Leishmania spp Real TM VER 21 03 2013 KEY TO SYMBOLS USED List Number LOT Lot Number Expiration Date Store at Manufacturer Consult instructions for use H E ra IC Internal Control 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 MX3005P6 is a registered trademark of Agilent Technologies ABI is a registered trademark of Applied Biosystems LineGeneK6 is a registered trademark of Bioer SmartCycler6 is a registered trademark of Cepheid Sacace Biotechnologies Srl VER NCA NCE C RUO Caution Contains sufficient for lt n gt tests Version Negative Control of Amplification Negative control of Extraction Positive Control of Amplification For Research Use Only via Scalabrini 44 22100 Como Italy Tel 390314892927 Fax 390314892926 mail info sacace com web www sacace com Sacace Leishmania s
5. M VER 21 03 2013 TROUBLESHOOTING 1 Weak or no signal of the IC FAM Green 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 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 JOE Yellow HEX Cy3 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 Repeat the DNA extraction with the new set of reagents 4 Any signal with Negative Control of P
6. _iSacace BIOTECHNOLOGIES Leishmania spp Real TM Handbook Real Time PCR kit for qualitative detection REF REF of Leishmania spp N3 50FRT TN3 50FRT NJ 50 ce Leishmania spp Real TM VER 21 03 2013 Sacace Leishmania spp Real TM VER 21 03 2013 NAME Leishmania spp Real TM INTRODUCTION Leishmaniasis is a parasitic disease that is found in parts of the tropics subtropics and southern Europe It is classified as a Neglected Tropical Disease NTD Leishmaniasis is caused by infection with Leishmania parasites which are spread by the bite of phlebotomine sand flies There are several different forms of leishmaniasis in people The most common forms are cutaneous leishmaniasis which causes skin sores and visceral leishmaniasis which affects several internal organs usually spleen liver and bone marrow Overall infection in people is caused by more than 20 species types of Leishmania parasites which are spread by about 30 species of phlebotomine sand flies The number of new cases per year is not known with certainty For cutaneous leishmaniasis estimates of the number of cases range from approximately 0 7 million to 1 2 million For visceral leishmaniasis estimates of the number of cases range from approximately 0 2 million to 0 4 million INTENDED USE Kit Leishmania spp Real TM is a test for the qualitative detection of Leishmania spp in the tissue specimens such as from skin so
7. a spp Real TM VER 21 03 2013 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 N metal tubing for reagent transfer Only for Module No 2 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 Leishmania spp Real TM can analyze DNA extracted from e tissue specimens from skin sores for cutaneous leishmaniasis e tissue specimens from bone marrow for visceral leishmaniasis Specimens can be stored at 2 8 C for no longer than 48 hours or frozen at 20 C to 80 C Transportation of clinical specimens must comply with country federal state and local regulations for the transport of etiologic agents Sacace Leishmania spp Real TM VER 21 03 2013 DNA ISOLATION The following kit is recommended gt DNA RNA Prep Sacace REF K 2 9 Please carry out DNA
8. ed 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 209 80 C Sacace Leishmania spp Real TM VER 21 03 2013 PROTOCOL Prepare required quantity of reaction tubes for samples N and controls N 2 Prepare in the new sterile tube for each sample 10 N 1 pl of PCR mix 1 FRT 5 0 N 1 of PCR Buffer FRT and 0 5 N 1 of TaqF DNA Polymerase Vortex and centrifuge for 2 3 sec Add to each tube 15 pl of Reaction Mix and 10 pl of extracted DNA sample to appropriate tube Mix by pipetting Prepare for each panel 2 controls e add 10 pl of DNA buffer to the tube labeled Amplification Negative Control e add 10 pl of Positive Leishmania spp C to the tube labeled Amplification Positive Control Insert the tubes in the thermalcycler Amplification Create a temperature profile on your instrument as follows Rotor type instruments Plate or modular
9. ely with plenty of water and seek medical advice 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 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 Sacace Leishmani
10. gh 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 Leishmania spp Real TM VER 21 03 2013 WARNINGS AND PRECAUTIONS The user should always pay attention to the following N X Component Lysis Sol 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 38 S 36 37 39 Risk Phrases R 20 21 22 Harmful by inhalation in contact with the skin and if swallowed R 22 Harmful if swallowed R 36 38 Irritating to eyes and skin Safety Phrases S 13 Keep away from food drink and animal feedstuffs N x Component Prec Sol contains 2 propanol flammable Irritant R10 36 67 S7 16 24 25 26 Risk Phrases R10 Flammable R36 37 38 Irritating to eyes respiratory system and skin R67 Vapors may cause drowsiness and dizziness Safety Phrases S7 Keep container tightly closed S16 Keep away from sources of ignition No smoking S24 25 Avoid contact with skin and eyes S26 In case of contact with eyes rinse immediat
11. n this case PCR should be repeated starting from the DNA extraction Boundary value of the cycle threshold Ct Ghannelitor Ct boundary value Sample Rotor type Plate type fluorophore s instruments instruments C4 FAM Green 30 30 JOE Yellow Hex Cy3 30 30 Clinical samples C FAM Green 30 30 Sacace Leishmania spp 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 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 SPECIFICATIONS Analytical sensitivity and reproducibility The analytical sensitivity of the Leishmania spp Real TM kit was determined using the Standard DNA of the Leishmania spp This Standard was serially diluted in the DNA buffer The analytical sensitivity of the kit Leishmania spp Real TM was not less than 1000 copies ml Sacace Leishmania spp Real T
12. pp Real TM VER 21 03 2013
13. res for cutaneous leishmaniasis or from bone marrow for visceral leishmaniasis PRINCIPLE OF ASSAY Kit Leishmania spp Real TM is based on two major processes isolation of DNA from specimens and Real Time amplification Leishmania spp DNA is extracted from the specimens amplified using Real Time amplification and detected by fluorescent reporter dye probes specific for Leishmania spp DNA and Internal Control Internal Control IC serves as an amplification control for each individually processed specimen and to identify possible reaction inhibition IC is detected in a channel other than the Leishmania spp Sacace Leishmania spp Real TM VER 21 03 2013 MATERIALS PROVIDED Module No 1 Real Time PCR kit N3 50FRT Part N 2 Leishmania spp Real TM Real Time amplification e PCR mix 1 FRT 0 6 ml e PCR Buffer FRT 0 3 ml e TaqF Polymerase 0 03 ml Pos Leishmania spp C 0 1 ml e Negative Control C 2 x 0 5 ml e internal Control IC 0 6 ml e DNA buffer 0 5 ml Contains reagents for 55 tests Module No 2 Complete Real Time PCR test with DNA purification kit TN3 50FRT Part N 1 DNA RNA Prep Sample preparation e Lysis Sol 15 ml e Prec Sol 20 ml e Washing Sol 3 25 0 ml e Washing Sol 4 10 0 ml e RE buffer 4 x 1 2 ml Contains reagents for 50 extractions Part N 2 Leishmania spp Real TM Real Time amplification e PCR mix 1 FRT 0 6 ml e PCR Buffer FRT 0 3
14. type instruments Step Temperature C Time Cycles Temperature C Time Cycles Hold 95 15 min 1 95 15 min 1 Cycling 95 5s 95 5s 1i 60 20s 5 60 20s 5 72 15s 72 15s 95 5s 95 5s Cycling 20s 30s 2 60 fluorescent 40 60 fluorescent 40 signal detection signal detection 72 15s 72 15s For example Rotor Gene 3000 6000 Q Corbett Research Qiagen 2 For example SaCycler 96 Sacace CFX iQ5 BioRad Mx3005P Agilent ABI amp 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 Leishmania spp is detected on the JOE Yellow HEX Cy3 channel C DNA on the FAM Green channel Sacace Leishmania spp Real TM VER 21 03 2013 NSTRUMENT SETTINGS Rotor type instruments RotorGene 3000 6000 RotorGene Q More Settings Channel Calibrate Gain Threshold Slope Correct Optimisation Outlier Removal FAM Green from 5 Fl to 10 FI 0 03 10 on JOE Yellow from 4 FI to 8 FI 0 05 10 on Plate or modular 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
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