Influenza A B Real TM Eng ver 21032013
Contents
1. If it is necessary to test less than 12 samples add for each sample N in the new sterile tube 10 N pl of RT G mix 1 with RT mix and 0 5 N pl of M MLV 2 Add 10 pl of Reaction Mix into each sample tube 4 5 Pipette 10 pl RNA samples to the appropriate tube If the Ribo Sorb isolation kit is used as a RNA extraction kit re centrifuge all the tubes with extracted RNA for 2 min at maximum speed 12000 16000 g and take carefully supernatant N B dont disturb the pellet sorbent inhibit reaction Carefully mix by pipetting Place tubes into thermalcycler and incubate at 37 C for 30 minutes Dilute 1 2 each obtained cDNA sample with TE buffer add 20 ul TE buffer to each tube cDNA specimens could be stored at 20 C for a week or at 70 C during a year Real Time amplification Reaction Mix 25 pl 1 2 Prepare required quantity of tubes or PCR plate Prepare for each sample in the new sterile tube 10 N pl of PCR mix 1 5 N pl of PCR mix 2 FRT and 0 5 N of TaqF Polymerase 3 Add 15 pl of Reaction Mix into each tube 4 Add10 pl of cDNA sample to appropriate tube with Reaction Mix Prepare for each panel 4 controls e add 10 pl of DNA buffer to the tube labeled Amplification Negative Control e add 10 ul of cDNA Influenza A C to the tube labeled Cos a e add 10 pl of cDNA Influenza B C to the tube labeled Cos 8 e add 10 ul of IC DNA to the tube labeled IC DNA55 Sacace Influenza A B Real TM VER 21 03 20132. Amplification 1 Create a temperature profile on your instrument as follows Rotor and plate type instruments Modular type instruments Step Temperature C Time Cycles Temperature C Time Cycles 1 95 15 min 1 95 900 s 1 95 10s 95 15s 25 S 2 54 25s 10 54 Fluorescence 42 detection 72 25s 72 25s 95 10s 30s 3 54 Fluorescence 35 detection 72 25 S For example SaCycler 96 Sacace CFX iQ5 BioRad Mx3005P Agilent ABI 7300 7500 StepOne Real Time PCR Applied Biosystems Rotor Gene 3000 6000 Q Corbett Research Qiagen LineGeneK Bioer For example SmartCycler Cepheid Influenza Virus A is detected on the Rox Orange channel Influenza Virus B is detected on the JOE Yellow channel C on the FAM Green channel INSTRUMENT SETTINGS Rotor type instruments Calibrate Gain More Settings Ema Optimisation Threshold Outlier Removal Sepo Teee FAM Green from 5 Fl to 10 FI 0 1 5 off JOE Yellow from 4 FI to 8 FI 0 1 10 off Rox Orange from 4 FI to 8 FI 0 1 10 off 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 samples Sacace
3. REAGENT PREPARATION reagents supplied with the module n0 2 1 10 11 12 13 14 15 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 Add to each tube 450 ul Lysis Solution and 10 pl Internal Control IC RNA Mix by pipetting and incubate 5 min at room temperature Add 100 ul of samples to the appropriate tube containing Lysis Solution and IC Prepare Controls as follows add 100 pl of C Negative Control to the tube labeled Cneg Vortex the tubes and centrifuge for 5 sec at 5000g If the sample is not completely dissolved it is recommended to re centrifuge the tube for 1 min at a maximum speed 12000 16000 g and transfer the supernatant into a new tube for RNA extraction Vortex vigorously Sorbent and add 25 pl to each tube Vortex for 5 7 sec and incubate all tubes for 10 min at room temperature Vortex periodically Centrifuge all tubes for 1 min at 10000g 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 tubes Add 400 pl of Washing Solution to each tube Vortex vigorously centrifuge for 1 min at 10000g and using a micropipette with a plugged aerosol barrier tip carefully remove and discard supern
4. sensitivity of not less than 500 copies ml Sacace Influenza A B Real TM VER 21 03 2013 TROUBLESHOOTING 1 Weak or absent signal of the IC Fam Green channel retesting of the sample is required e The PCR was inhibited gt Make sure that you use a recommended RNA extraction method and follow the manufacturer s instructions gt Re centrifuge all the tubes before pipetting the extracted RNA 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 for the IC detection select 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 RNA extraction procedure 2 Weak signal on the Joe Yellow Cy3 HEX and Rox TexasRed channels retesting of the sample is required 3 Joe Yellow Cy3 HEX or Rox TexasRed signal with Negative Control of extraction e Contamination during RNA extraction procedure All samples results are invalid Decontaminate all surfaces and instruments with sodium hypochlorite and ethanol gt Use only filter tips during the extraction procedure Change tips among tubes Repeat the RNA extraction with the new set of reagents 4 Any signal with Negative PCR Contro
5. x g Eppendorf 5415D or equivalent e 60 C 2 C dry heat block e Vortex mixer e Pipettors capacity 5 40 ul 40 200 pl 200 1000 ul with aerosol barrier e 1 5 ml polypropylene sterile tubes Sarstedt QSP Eppendorf e Disposable gloves powderless e Tube racks e 70 Ethanol freshly prepared mixture of reagent grade 96 ethanol and distilled water e Acetone e Refrigerator e Freezer Zone 2 RT and amplification e Real Time Thermalcycler e Workstation e Pipettors capacity 0 5 10 ul 5 40 pl with aerosol barrier e Tube racks STORAGE INSTRUCTIONS Influenza Virus A B Real TM must be stored at 20 C Store Ribo Sorb kit at 2 25 C The kits can be shipped at 2 8 C for 3 4 days but should be stored at 2 8 C and 20 C immediately on receipt STABILITY Influenza Virus A B 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 Influenza A B Real TM VER 21 03 2013 WARNINGS AND PRECAUTIONS In Vitro Diagnost
6. Influenza A B Real TM VER 21 03 2013 RESULTS ANALYSIS 1 The sample is considered to be positive for Influenza Virus A if in the channel Rox Orange the value of Ct is different from zero must be less than 33 37 for SmartCycler If Ct value is more than 33 the assay should be repeated and the sample is considered to be positive in case of result s repeat or in case of result is less than 33 2 The sample is considered to be positive for Influenza Virus B if in the channel Joe Yellow the value of Ct is different from zero must be less than 33 37 for SmartCycler If Ct value is more than 33 37 for SmartCycler the assay should be repeated and the sample is considered to be positive in case of result s repeat or in case of result is less than 33 3 The sample is considered to be negative for Influenza A B if in the channel Fam Green value is not determined the fluorescence curve does not cross the threshold line and in the results table on the channel Fam Green the Ct value is lower than 28 Table Results for controls Stage for NCE ei Pos lt 28 Neg Neg Valid result NCA PCR Neg Neg Neg Valid result Pos cDNA Infl A PCR Neg Neg Pos lt 33 Valid result Pos cDNA Infl B PCR Neg Pos lt 33 Neg Valid result IC DNA PCR Pos lt 28 Neg Neg Valid result PERFORMANCE CHARACTERISTICS The kit Influenza A B Real TM allows to detect nfluenza A amp B viruses in 100 of the tests with a
7. _lSacace BIOTECHNOLOGIES w VD For in Vitro Diagnostic Use CE Influenza A B Real TM Handbook Real Time Amplification test for the detection of Influenza A and B Viruses REF V36 50FRT REF TV36 50FRT amp 50 Sacace Influenza A B Real TM VER 21 03 2013 NAME Influenza Virus A B Real TM INTRODUCTION Influenza virus infection one of the most common infectious disease is a highly contagious airborne disease that causes an acute febrile illness and results in variable degrees of systemic symptoms ranging from mild fatigue to respiratory failure and death These symptoms contribute to significant loss of workdays human suffering mortality and significant morbidity Influenza results from infection with 1 of 3 basic types of influenza virus A B or C which are classified within the family Orthomyxoviridae These single stranded RNA viruses are structurally and biologically similar but vary antigenically The most common prevailing influenza A subtypes that infect humans are H1N1 and H3N2 INTENDED USE Influenza Virus A B Real TM is Real Time amplification test for the qualitative detection of Influenza A and B RNA in clinical specimens PRINCIPLE OF ASSAY Influenza Virus A B Real TM Test is based on three major processes isolation of virus RNA from specimens reverse transcription of the RNA Real Time amplification of the cDNA Influenza virus A amp B detection by the polymerase chai
8. atant from each tube without disturbing the pellet Change tips between tubes Add 500 pl of Ethanol 70 to each tube Vortex vigorously centrifuge for 1 min at 10000g 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 tubes Repeat step 10 Add 400 pl of Acetone to each tube Vortex vigorously centrifuge for 1 min at 10000g 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 tubes Incubate all tubes with open cap for 10 min at 60 C Resuspend the pellet in 40 pi of RNA eluent Incubate for 10 min at 60 C and vortex periodically Centrifuge the tubes for 2 min at maximum speed 12000 16000 g The supernatant contains RNA ready for use The RT PCR can be performed the same day If this is not possible the RNA preparations can be stored at 80 C for up to one month Sacace Influenza A B Real TM VER 21 03 2013 RT AND AMPLIFICATION Reverse Transcription 1 Prepare Reaction Mix for 12 reactions add 5 0 pl RT G mix 1 into the tube containing RT mix and vortex for at least 5 10 seconds centrifuge briefly This mix is stable for 1 month at 20 C Add 6 pl M MLV into the tube with Reagent Mix mix by pipetting vortex for 3 sec centrifuge for 5 7 sec must be used immediately after the preparation
9. chlorite 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 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 Influenza A B 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 Influenza Virus A B Real TM can analyze RNA extracted with Ribo Sorb REF K 2 1 from e nasopharyngeal swabs swab area and place in Eppendorf tube with 0 5 ml
10. ic Medical Device 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 Clinical specimens from suspect influenza A H1N1 cases should be performed in a BSL2 laboratory with BSL3 practices enhanced BSL2 conditions 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 hypo
11. l 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 controls at the end Repeat the PCR preparation with the new set of reagents Sacace Influenza A B Real TM VER 21 03 2013 Sacace Influenza A B Real TM VER 21 03 2013 Sacace Influenza A B Real TM VER 21 03 2013 KEY TO SYMBOLS USED List Number Caution LOT LSE N nbei ii sufficient Or lt n gt tests IVD de in Vitro Diagnostic VER Version se Store at NCA Negative CONtorof 1 Amplification ual Manufacturer NCE St conttolol xtraction li Consult instructions for Positive Control of C pag use Amplification po Expiration Date IC Internal Control SaCycler is a registered trademark of Sacace Biotechnologies CFXTM 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 via Scalabrini 44 22100 Como Italy Tel 390314892927 Fax 390314892926 mail info sacace com web www sacace com Sacace Influenza A B Real TM VER 21 03 2013
12. n reaction PCR is based on the amplification of pathogen genome specific region using specific primers and detection via fluorescent dyes These dyes are linked with probes of oligonucleotides which bind specifically to the amplified product The real time PCR monitoring of fluorescence intensities allows the accumulating product detection without reopening of reaction tubes after the PCR run Influenza Virus A B Real TM PCR kit is a qualitative test which contain the Internal Control IC It must be used in the isolation procedure in order to control the process of each individual sample extraction and serves also to identify possible reaction inhibition Sacace Influenza A B Real TM VER 21 03 2013 MATERIALS PROVIDED Module No 1 Real Time PCR kit V36 50FRT Part N 2 Reverta L Reverse transcription of the RNA e RT G mix 1 5 x 0 01 ml e RT mix 5 x 0 125 ml e Reverse transcriptase M MLV 0 03 ml e TE buffer 1 2 ml Contains reagents for 60 tests Part N 3 Influenza A amp B Real Time amplification kit e PCR mix 1 5 x 0 12 ml e PCR mix 2 FRT 0 3 ml e TaqF Polymerase 0 03 ml e Pos cDNA Infl A C 0 1 ml e Pos cDNA Infl B C 0 1 ml e Negative Control 1 2 ml e Internal Control RNA 5 x 0 12 ml e Internal Control DNA 0 1 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 Inte
13. of saline water or PBS sterile Sacace Transport medium is recommended Agitate vigorously Repeat the swab and agitate in the same tube Centrifuge at 1000g min for 5 min Discard the supernatant and leave about 100 ul of solution for RNA extraction e aspirate bronchial lavage nasal wash centrifuge at 2000 g min for 10 15 min If the pellet is not visible add 10 ml of liquid and repeat centrifugation Remove and discard the supernatant Resuspend the pellet in 100 pl of Saline water e tissue 1 0 gr parenchimatous organs trachea lung brain homogenized with mechanical homogenizer or scalpel glass sticks teflon pestles and dissolved in 1 0 ml of saline water or PBS sterile Vortex vigorously and incubate 30 min at room temperature Transfer the supernatant into a new 1 5 ml tube Specimens can be stored at 2 8 C for no longer than 12 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 RNA ISOLATION The following isolation kits are recommended gt Ribo Sorb Sacace REF K 2 1 Ribo Virus spin column extraction kit Sacace REF K 2 C Please carry out the RNA extraction according to the manufacturer s instructions Add 10 ul of Internal Control during the DNA isolation procedure directly to the sample lysis mixture Sacace Influenza A B Real TM VER 21 03 2013 SPECIMEN AND
14. rnal Control RNA during the RNA purification procedure directly to the sample lysis mixture Sacace Influenza A B Real TM VER 21 03 2013 Module No 2 Complete Real Time PCR test with RNA purification kit TV36 50FRT Part N 1 Ribo Sorb Sample preparation e Lysis Solution 22 5 ml e Washing Solution 20 ml e Sorbent 1 25 ml e RNA eluent 5 x 0 5ml Contains reagents for 50 tests Part N 2 Reverta L Reverse transcription of the RNA e RT G mix 1 5 x 0 01 ml e RT mix 5 x 0 125 ml e Reverse transcriptase M MLV 0 03 ml e TE buffer 1 2 ml Contains reagents for 60 tests Part N 3 Influenza A amp B Real Time amplification kit e PCR mix 1 5 x 0 12 ml e PCR mix 2 FRT 0 3 ml e TaqF Polymerase 0 03 ml e Pos cDNA Infl A C 0 1 ml e Pos cDNA Infl B C 0 1 ml e Negative Control 1 2 ml e Internal Control RNA 5 x 0 12 ml e Internal Control DNA 0 1 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 RNA during the RNA purification procedure directly to the sample lysis mixture Sacace Influenza A B Real TM VER 21 03 2013 MATERIALS REQUIRED BUT NOT PROVIDED Zone 1 sample preparation only for Module No 2 e RNA extraction kit Module No 1 e Biosafety cabinet e Desktop microcentrifuge for eppendorf type tubes RCF max 16 000
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