HIV RNA Real TM 100 PCR NEW
Contents
1. Target C Hold hh mm ss pum Rate C s Sec Target Step Size C C 95 C 0 20 None 55 C 0 40 5 s None 72 C 0 30 Step Delay Acquisition Mode cycles E None Acquisition mode Single Ib 52 00 00 30 None fe 00 00 30 None LA Program 40 m Quantification gt Target C Hold hh mm ss Ramp Rate C s Step Delay Acquisition Mode cycles g5 00 00 20 10 None E 55 00 00 30 10 Single E 72 00 00 30 10 None M 0 00 00 0 30 02 0 45 09 1 00 17 215737 1 30 40 1 45 56 Time h mm ss Temperature C no O 3 Click and insert sample names then select Absolute Quantification from Analysis Type and select 530 560 in Selected Channels section Sample data Analysis Type j Reset Samples Impc gi Absolute Quantification Color Compensation Genotyping Nucleic Acid Quantification Qualitative Detection Relative Quantification Dual Color Relative Quantification Monacolor Tm Calling imi T Tel ln en Selected Channels 530 560 610 64 4 Insert target Name Sample Type Standard for standards Unknown for all other samples and insert sample concentration values as reported in the Data Card 5 Click gtatfn to start PCR reaction 6 After Run is completed click Analysis button and select Absolute quantif2. 5 t Blank Control Control Next replicate number is gt 7 Per Dye Layer ies v Scientific Notation Define sample C3 Standard Quantity Sample Identifier and replicates m m Running Instrument not in Remote 14 11 2006 16 11 Click Select and Load Fluorophores in the Edit Plate Setup window and select Joe 530 and FAM 490 Double click in the Plate Setup Filename field at the top left of the window and enter a name for the plate setup file and click Save this plate setup Click Run with selected protocol Indicate reaction volume 25 ul Select PCR Quantification Melt Curve and Experimental Plate Click Begin Run DATA ANALYSIS e Click View Post Run Data in the top right of Library module Select the name of Data file and click Analyze Data e Select PCR Quantification window of Data Analysis module and set icon JOE 530 in Select a Reporter Click User Defined near the displayed threshold position and enter 50 into the Threshold Position field Click Recalculate Threshold Cycles Make sure that in the window Select Analysis Mode is selected PCR Baseline Subtracted Curve Fit Select PCR Standard Curve tab In the new window appear curves and values of HIV cDNA e Make a similar procedure for FAM 490 channel Click User Defined near the dis
3. Decontaminate all surfaces and instruments with sodium hypochlorite and ethanol Use only filter tips during the extraction procedure Change tips among tubes Repeat the RNA extraction with the new set of reagents 6 Any signal with Negative PCR Control e Contamination during PCR preparation procedure All samples results are invalid gt 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 EXPLANATION OF SYMBOLS Catalogue Number For Research Use Only Lot Number d Expiration Date Ny Contains reagents N Caution Version T Manufacturer Temperature limitation Cycler and 1Q5TM are trademarks of Bio Rad Laboratories Rotor Gene Technology is a registered trademark of Corbett Research MX3000P and MX3005P are trademarks of Stratagene Applied Biosystems is trademarks of Applera Corporation LightCycler is a registered trademark of Roche LineGene K is a registered trademark of Bioer Eco PCR Real Time System is a registered trademark of Illumina Sacace Biotechnologies Srl via Scalabrini 44 22100 Como Italy Tel 390314892927 Fax 390314892926 mail info sacace com web www sacace com e C RM T SISTEMA SISTEMA ESTIONE DI GESTI CERTIFICATO 13485 2004 DI GESTIONE CERTIFICAT
4. 0 025 mL o QS31C 4x 0 025 mL Standards and controls concentrations are specific for every lot MATERIALS REQUIRED BUT NOT PROVIDED RNA isolation kit see RNA isolation Desktop microcentrifuge for eppendorf type tubes Vortex mixer Disposable gloves powderless Biohazard waste container Refrigerator Freezer Real Time Thermal cycler Workstation Pipettes adjustable Sterile pipette tips with filters Tube racks WARNINGS AND PRECAUTIONS l pm ere O OND NM Bf W NO pec m cm ue c cm couRo c c9 S d Un 4 GW Wear disposable gloves laboratory coats and eye protection when handling specimens and reagents Thoroughly wash hands afterward Do not pipette by mouth Do not eat drink smoke apply cosmetics or handle contact lenses in laboratory work areas Do not use a kit after its expiration date Do not mix reagents from different kits Biosafety Level 2 should be used for materials that contain or are suspected of containing infectious agents Specimens and controls should be prepared in a laminar flow hood Heparin has been shown to inhibit reaction Use of heparinized specimens is not recommended Avoid repeated thawing and freezing of the reagents this may reduce the sensitivity of the test Once the reagents have been thawed vortex and centrifuge briefly the tubes Prepare quickly the Reaction mix on ice or in the cooling block Specimens may be infectious Use Universal Preca
5. HIV Real TM Quant Anril 12 ON 1 JCOce HIV Real TM Quant API 1o ZU 10 11 12 L5 Apply Analysis Settings Click Apply y g Select all samples and standards click View well table and then Click File gt Export select Results only Sample Setup 7 Results Raw Data Multicomponent Data mplif tion Data Export Properties 1 Select data to export Click Customize Export and sort samples by reporter as indicated in the above image Then click uem i export results in a file Using the exported file calculate results pasting data from the Sample and the Quantity columns in the provided HIV Quant Result Calculation sheet LightCycler 2 048 Roche l Select New LightCycler Experiment Click and insert as follows Setup Default Channel 530 e Max Seek Pos indicates the number of samples used in the experiment Seek Temperature 30 Choose Instrument Type according to your instrument ba a Max Seek Pos 19 Instrument Type 6 Ch Choose capillary Size according to your capillaries we Capillary Size 20 ul suggest to select 20 ul and use 30 ul polycarbonate capillaries ENEN 2 Program the thermalcycling as follows HIV Real TM Quant Lll Program gione Stage Step Delay Acquisition Mode 1 50 C 30 00 ees 2 95 C 15 00 Reps 50 00 30 00 410 0 u zi None 95 C 0 20 5 52 C 0 30 ESTA EIN 72 C 0 30 gra lt None o em
6. Selected Plate Setup Template pts All changes must be saved before running Selected Plate Setup is loaded in the Edit Plate Setup tab Show options Select data collection step s 20 Step 2 REAL TIME Ll Step 3 1 1 Cycle Repeats Step peel Setpoint es TERR ae Time 1 1 30 00 50 0 J 1 15 00 z 1 00 20 2 00 30 3 00 30 45 1 00 30 2 00 40 3 00 30 Double click in the Protocol File Name text box and enter a new name for the protocol Click Save this protocol Select Edit Plate Setup to create the plate for samples and standards In the new window click Samples Whole Plate loading Use icon Unknown for the wells that contain samples icon Standard to identify wells that contain calibrators and Control for Negative Control Give a name for the samples in the Sample Identifier box Enter any name for the standard in the Identifier box and enter the concentrations of the Quantitative Standards reported on the HIV TM 1Q Quant Data Card 18 xi Edit Protocol Edit Plate Setup View Quantities Identifiers Run Prep Save this plate setup Selected Protocol is loaded in the Edit Protocol tab All changes must be saved before running Run with selected protocol mi Click a sample icon for loading wells roO OU Standard Unknown Samples Whole Plate loading Select and load fluorophores Click Cursor to view a well s content
7. each tube 5 Add12 5 ul of extracted RNA sample to the appropriate tube with Reaction Mix and mix by pipetting 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 don t disturb the pellet sorbent inhibit reaction 6 Prepare for each run 6 standards and 1 negative control e add 12 5 ul of Quantitation Standards HIV QS1 HIV QS2 HIV QS3 HIV into 3 labeled tubes e add12 5 ul of Quantitation Standards IC QS1 IC QS2 IC QS3 IC into 3 labeled tubes e add 12 5 ul of TE buffer to the tube labeled Negative Control PROTOCOL DATA ANALYSIS RotorGene M 3000 6000 Q Corbett Reasearch Qiagen 1 Close tubes and transfer them into the carousel of the Rotor Gene 3000 6000 Q 2 Click New in the main menu 3 Select New Run and Dual Labeled Probe Click New LEN Ex Quick Start Advanced Perform Last Run A SYBR Green R I al Labeled Probe Quenched FRET DN Concentration Measurement A Hybridization A Melt v Show this Screen when Rotor Gene Opens 7 Rotor Gene Demo Kit gt Open Template In Another Folder Cancel New Help Program Rotor Gene 2000 3000 6000 as follows gt Select Rotor Type 36 Well Rotor and No Domed 0 2 ml Tubes gt Reaction Volume ul 25 Temperature Profile 1 Hold 50 C 30 min 2 D
8. 1 A2 Posi 1 A3 Pos2 1 A4 QSL HIV 1 S 2 35e 004 A5 QS2HIV 1 S 1 35e 003 A6 QS3HIV 1 S 6 60e 001 A7 QSiIC 1 gt S 327e 004 A8 QS2IC 1 S 1 60e 003 A9 QS3IC 1 S 6 40e 001 A10 NEGPCR 1 All 1 4 Click or click File gt Run Programs R to Run the PCR program Results Analysis FAM In the Quantitative Analysis tab select from the menu Dyes D gt FAM F Select standard Concentration boxes left mouse click CTRL for Internal Control see picture below Concentration SN Group Sample A 1 Neg Extr A 1 Posi A3 1 Pos2 M 1 QS1 HV WC 1 QS2HIV 6 1 QS3 HIV M 1 QSLIC A8 1 QS21C 9 1 QS3IC Alo 1 NEG PCR 2 35e 004 1 35e 003 6 60e 001 Then do in this order 1 Under Analysis Method select Fit Points 2 Under Analysis Step Click Zero Adjust select Auto and then click OK Then click Baseline 3 Select 2 points and set the base line drag and drop the red baseline as low as possible but above the noise of each sample usually 10 12 4 Under Analysis Step clic Analysis Calculated copies reaction of Internal Control and Ct Results should appear in the Ct and the Calculated Concentration column respectively Analysis Method 2nd Driv Max MIFit Points Results Analysis Analysis Step Points Selected Zero Adjust M Baseline E Analysis El Show Points Joe Baseline and Threshold Display Mode El Logar
9. Identifier a 18007 1800 Cycle i 1600 1600 c4 2 26 6 16 t SS cs g 27 3 M10 2 1401 piso y 27 5 f115 a 26 4 M8 Run Time x 1 L c 24 2 H105 3 Central ps f m s 26 7 C10 J 1200 A T 1 c 26 3 H118 5 Y j c7 8 27 7 B6 1000 Ad i i c 26 8 L75 a 27 1 M13 3 Yor 19 3 L96 26 3 24 j x j a c9 24 7 L45 8 26 3 F4 C10 24 0 12 E 26 7 PKO1 1 ps 24 247 z 27 1 PK02 a 400 4 31 9 PKO1 26 8 OKO A g 1 D4 N A K1 Analpsis a ix 25 9 PKO2 T T D5 N A OKO 1004 00 N A K2 1 De 18 5 K1 es ua n 1 sons de i N U SS DS OS a oe s 200 a 25 9 K2 3 ans D7 26 4 B2 0 4 12 14 16 18 20 4 4 42 44 33 9 K3 E N A BL aij 2 N A 82 ACTION Data Window Last 95 Filter Weighted Mean Analysis Post Run ACTION Data Window Last 95 Filter Weighted Mean Analysis Post Run Running Instrument not in Remote 05 12 2006 15 15 e Select PCR Standard Curve tab In the new window appear curves and values of IC cDNA In results table PCR Standard Curve appear Ct Threshold cycle values which should be lt 28 if value is more than 28 a retesting of sample is required e The Coefficient correlation value R in Standard Curve window should be 0 9 if 0 9 a retesting of all samples is required e Ifthe Ct value of the specimen is higher than 40 a retesting of the sample is required V Applied Biosystems 7300 7500 StepOne Real Time PCR Systems Applera 1 Select in the main menu
10. Name HIV Real TM Quant e El Hotlid C Liquid Quant ul Click the button 997 4 times and then program the 4 thermalcycling steps as in the following table Target Temp C Incubation Time eee Transition Rate C s Mode Cycle Temp 1 HI i 03000 00 None On the second phase 55 0 C of step 4 select Single on the Sample Mode column to collect fluorescence data as shown in this picture Target Time Rate 2nd Temp Step Size Step Delay Grad Temp Sample Mode 95 0 00 00 20 None E ME MEM MNNCENNE 72 0 00 00 30 ee Click the menu System Parameters P gt Gain Setup G Select Auto Gain box and click OK see picture below for Gain Setup details FEWEST Groups G Dye m Fitted Data F Raw Data R owes 78 E 2 E BEEE wwe 30 99 Ce C Ed Auto Gain f o o gt f cancel C Grad Temp Calculation C Gain Setup G Select System Parameters S Crosstalk Measurement R Crosstalk Dyes Measured T Click the menu Sample Information S gt sample Group Setup R Select at least 2 groups Click OK AT Sample Group Setup Click the menu Sample Information S gt Sample Data S Insert Sample name Property U for unknown samples and negative PCR control S for Standards and insert standard concentration reported in Data Card Group Type Concentration A Neg Extr
11. O 9001 2008 PCR The Polymerase Chain Reaction PCR process is covered by patents owned by Hoffmann La Roche and applicable in certain countries Sacace does not encourage or support the unauthorized or unlicensed use of the PCR process Use of this kit is recommended for persons that either have a license to perform PCR or are not required to obtain a license
12. RNA DNA extraction kit Sacace K 2 16 1000 Please carry out the RNA extraction according to the manufacturer s instructions Add 5 ul of Internal Control during the RNA isolation procedure directly to the sample lysis mixture see Internal Control INTERNAL CONTROL HIV Rec IC HIV Rec IC is a quantitative Internal Control concentration reported in Data Card and represents recombinant RNA containing structure which carried through all steps of analysis from nucleic acid extraction to PCR amplification detection The presence of quantitative HIV Rec IC allows not only to monitor the extraction procedure and to check possible PCR inhibition but also to verify possible losses of the RNA during extraction procedure thus enabling to calculate precisely the HIV viral load Protocol 1 Thaw one set of reagents vortex and centrifuge briefly the tubes 2 Prepare 0 2 ul tubes or PCR plate 3 Prepare Reaction Mix add into the tube with DTT 300 ul of RT PCR mix 1 HIV 200 pl of RT PCR mix 2 20 ul of Hot Start TaqF Polymerase and 7 5 ul of M MLV Revertase Vortex thoroughly and centrifuge briefly If it is necessary to test less than 25 samples add into the tube with DTT 300 ul of RT PCR mix 1 200 ul of RT PCR mix 2 and vortex for at least 5 10 seconds This mix is stable for 1 month at 20 C Add for each sample N in the new sterile tube 12 5 N wl of mix 0 5 N ul of TaqF Polymerase and 0 25 N ul of M MLV 4 Add 12 5 ul of Reaction Mix into
13. _tSacace BIOTECHNOLOGIES HIV Real TM Quant Real Time test for the quantitative detection of Human Immunodeficiency Virus HIV RNA for use with RotorGene 3000 6000 Q Corbett Research Qiagen 1Q 1CyclerTM and 1Q5 Biorad MX3000P and MX3005P Stratagene Applied Biosystems 7300 7500 StepOne Real Time PCR Systems Applera LightCycler 2 0 Roche LineGene K Bioer Eco Real Time PCR System Illumina R VM 100 FRT Sacace HIV Real TM Quant April 18 2011 NAME HIV Real TM Quant INTRODUCTION HIV is a lentivirus a member of the retrovirus family differentiated on structural and antigenic properties into two virus types HIV 1 and HIV 2 HIV 2 occurs considerably less often than HIV 1 In accordance with 1991 Nomenclature there are three independent HIV 1 groups M main O outlier N non V non O In addition to this there are the so called circulating recombinant forms CRF viruses with a mosaic structure of the genome elements of which are typical for representatives of various subtypes Groups O and N are less widely spread and occur in African countries population Group M includes 11 subtypes Al A2 B C D F1 F2 G H J K Transmission ways of the virus are very important for the virus spread HIV is transmitted by three ways at heterosexual and homosexual sexual intercourse parenteral with blood and blood products and vertically from the infected mother to the child by an intrauterine
14. eic acid extraction to PCR amplification detection The presence of quantitative HIV Rec IC allows not only to monitor the extraction procedure and to check possible PCR inhibition but also to verify possible losses of the RNA during extraction procedure thus enabling to calculate precisely the HIV viral load Monitoring the fluorescence intensities during Real Time allows the detection and quantification of the accumulating product without having to re open the reaction tube after the real time amplification The kit will allow the quantification of 100 samples including all the necessary reagents to generate the external standard curve of HIV and IC To generate HIV and IC standard curve for quantification of the amplification products all calibrators should be used and defined as standards with specific concentrations MATERIALS PROVIDED Part N 1 Controls Part N 2 HIV Real TM Quant RT Real Time kit Part N 1 Controls HIV Rec Pos Control 4 x 0 01 ml HIV Rec Pos2 Control 4 x 0 01 ml Negative Control 4 x 0 5 ml HIV Rec IC Internal Control 4 x 0 13 ml Part N 2 HIV Real TM Quant DTT 4 tubes RT PCR mix 1 TM HIV 4 x 0 3 mL RT PCR mix 2 TM 4 x 0 2 mL Hot Start TaqF Polymerase 4 x 0 02 mL M MLV Revertase 4 x 0 01 mL TE buffer 4 x 0 07 mL Standard HIV o QSIHIV 4 x 0 025 mL o QS2HIV 4 x 0 025 mL o QS3HIV 4 x 0 025 mL Standard IC o QSIIC 4x 0 025 mL o QS2IC 4x
15. enature 95 C 15 min 3 Cycling 95 C 20 s 52 C 30s 72 C 30s Cycle repeats 5 times 4 Cycling 95 C 20s 55 C 30 s 72 C 30s Cycle repeats 40 times Fluorescence is measured at 55 C on FAM Green and JOE Yellow channels In the window New Run Wizard click Calibrate Gain Optimisation for Rotor Gene 6000 In the new window Channel Setting select channels Joe Yellow and Fam Green Indicate Tube Position 1 Min Reading 5 Max Reading 10 and select function Perform calibration Optimization before 1 Acquisition Click Close button Select Next and click Start run Program position of the tubes in the carousel of the Rotor Gene 2000 3000 6000 and enter the concentrations of the Quantitative Standards reported on the HIV Real TM Quant Data Card in order to generate Standard curve Results Analysis IC amplification analysis channel Cycling A Fam Green l 2 3 4 Press Analysis then select Quantitation Cycling A Fam Cycling A Green Show Turn off the automatic option Threshold Press buttons Dynamic Tube Slope Correct Select Threshold 0 03 In the table of results Quantitation Results appear the values of Ct Threshold cycle which should be x 28 If the Ct value of the specimen is absent or higher than 28 a retesting of the sample is required In the menu window Quantitation Results column Calculation concentration appear values of IC cDNA copies specimen The Coefficient correlat
16. g the following formula HIV DNA copies specimen IC DNA copies specimen coefficient is specific for each lot and reported in the HIV Real TM Quant Data Card provided in the kit Results may by calculated also using HIV Quant Result Calculation sheet provided with the kit x coefficient copies HIV mL HIV Real TM Quant is linear from 25 to 5 x 10 copies mL Test results greater than 5 000 000 copies mL are above the upper limit of quantitation of the test and should be reported as greater than 5 000 000 copies mL If quantitation results are desired for such samples the specimen should be diluted 1 10 with negative serum and retested Test results less than 25 copies mL are below the lower limit of quantitation of the test and should be reported as less than 25 copies mL PERFORMANCE CHARACTERISTICS Analytical specificity The analytical specificity of the primers and probes was validated with negative samples They did not generate any signal with the specific HIV primers and probes The specificity of the kit HIV Real TM Quant was 100 The potential cross reactivity of the kit HIV Real TM Quant was tested against the group control It was not observed any cross reactivity with other pathogens Analytical sensitivity The kit HIV Real TM Quant allows to detect HIV RNA in 100 of the tests with a sensitivity of not less than 25 copies ml The detection was carried out on the control standard and its dilutions by negative
17. ication Select Method Auto Standard Curve In Run Ct values and calculated results will appear for the selected channel under the CP and Concentration column in the Absolute Quantification table 7 Calculate results manually using the usual formula as indicated in the User Manual of the kit A At first usage the operator must perform a color compensation experiment asking Sacace for the detailed instructions Eco Real Time PCR System Illumina lai Set Up Standards Gim uantification Hydrolysis Probe Open Eco software click experiment and under Experiment Type select T MUR select RNA as starting material for HIV Real TM Quant U i o Standard Curve l nder Quantification Method select and insert experiment name Click Click Q set up two assays one for HIV Reporter HEX and the other one for Internal Control Reporter Fam select Quencher Non fluorescent for both targets Click Click and enter name for all samples controls and standards Click Associate samples and standards with the two previously designed assays following this procedure select the well select Unknown for samples and Standard for standards and to assign the corresponding assay click the white circle of the HCV and Internal control assays the circle will become colored for each well both Assays must be assigned Click d and in the Set Up Standards box insert each value reported in the DataCard click Enter after insert
18. ing and freezing of these reagents should be avoided as this may reduce the sensitivity Components stored under conditions other than those stated on the labels may not perform properly and may adversely affect the assay results SAMPLE COLLECTION STORAGE AND TRANSPORT Note Handle all specimens as if they are potentially infectious agents l EDTA tubes may be used with the HIV Real TM Quant Follow sample tube manufacturer s instructions 2 Whole blood collected in EDTA should be separated into plasma and cellular components by centrifugation at 800 1600 x g for 20 min within six hours The isolated plasma has to be transferred into a sterile polypropylene tube Plasma may be stored at 2 8 C for an additional 3 days Alternatively plasma may be stored at 18 C for up to one month or year when stored at 70 C Do not freeze whole blood Specimens anti coagulated with heparin are unsuitable for this test Thaw frozen specimens at room temperature before using Whole blood must be transported at 2 25 C and processed within 6 hours of collection Plasma may be transported at 2 8 C or frozen 7 Transportation of clinical specimens must comply with country federal state and local regulations for the transport of etiologic agents Nn 4 YW RNA ISOLATION The following isolation kits are recommended Ribo Virus 100 spin column extraction kit Sacace K 2 C 100 Ribo Sorb 100 Sacace REF K 2 1 100 Magno Sorb Magnetic
19. ing each value both for HIV assay and Internal control assay then associate concentration values to the corresponding well In the end it should look like above s Define Standards W L Units copies u v vu Assign Quantity i 68350 Sampe4 MSp Sample 6 Sampe SSP SampeS9 8220 goo ET 9 TY Y W W 760 B NU Wy NU Ney 88250 ud wu uY d 10 11 Stage 0 Click on mune and program the instrument as follows reaction volume is 20 ul HIV Real TM Quant Reps Add 10 ul of Reaction Mix into 50 C 30 00 each tube 95 C 15 00 Add 10 wl of extracted RNA sample or quantitative standards to the 95 C 0 20 appropriate tube with Reaction Mix 52 C 0 30 72 C 0 30 72 C 0 30 95 C 0 20 OM _ AN 55 C 0 40 40 Click Start Run to begin PCR reaction a Amplification Plot After the run is complete click then click Ns tab to interpret the results Click Y to Export the samples in the Export Options select Results Table Using the exported file calculate results pasting data from the Sample Name and the Quantity columns in the provided HIV Quant Result Calculation sheet LineGene K Bioer Open LineGeneK Software Click File New In the Setup Programs tab Insert User Name and Test Name Click Dyes button and select FAM JOE Insert the correct liquid quantity 25 ul ier Nane Test
20. ion value R in the Standard Curve window should be 0 9 if 0 9 a retesting of all samples is required HIV amplification analysis channel Cycling A Joe Yellow l PA 3 4 GN Press Analysis then select Quantitation Cycling A Joe Cycling A Yellow Show Turn off the automatic option Threshold Press buttons Dynamic Tube Slope Correct In the table of results Quantitation Analisis select More settings for VI version of software or Quant Settings for V version Set NTC Threshold to 5 Select Threshold 0 03 In the table of results Quantitation Analisis appear the values of Ct Threshold cycle In the menu window Quantitation Results appear values of HIV cDNA copies specimen If the Ct value of the specimen is higher than 40 a retesting of the sample is required The Coefficient correlation value R in Standard Curve window should be 0 9 if lt 0 9 a retesting of all samples is required iQ iCycler and iQ5 Biorad Select in the main menu Define Protocols and click Create a new protocol Set the following parameters Set Point 50 0 95 0 95 0 52 0 72 0 95 0 S5 0 72 0 30 00 Fluorescence is measured at 55 C on FAM and JOE HEX channels DR Services Help in the Standard Quantity box E erer K lBix Edit Plate Setup View Quantities Identifiers Save this protocol 4 gt Protocol Filename RNA HIV FRT tmo
21. ithmic EFull Scale Smooth Low In the Quantitative Analysis tab select from the menu Dyes D gt Joe F Select standard Concentration boxes left mouse click CTRL for HIV Group Sample Concentration Al 1 Neg Extr iw 1 Posi NES 1 Pos2 m E E 20 ss B lone x WEM ov 1 m 1 QSLIC 327e 004 5 enn 1 Qs2IC 1 60e 003 1 QS3IC 6 40e 001 AO 1 NEG PCR Then do in this order 1 Under Analysis Method select Fit Points 2 Under Analysis Step Click Zero Adjust select Auto and then click OK Then click Baseline 3 Select 2 points and set the base line drag and drop the red baseline as low as possible but above the noise of each sample usually 10 15 4 Under Analysis Step clic Analysis Calculated copies reaction of HIV and Ct Results should appear in the Ct and the Calculated Concentration column respectively Analysis Method Analysis Step Points Selected Baseline and Threshold EJ2nd Driv Max Zero Adjust Baseline E Baseline EJFit Points ElAnalyss show Points Threshold Internal control FAM 70 8 8 Fluorescence s T Display Mode e Logarithmic Full Scale Smooth Low Ed HIV RNA Joe 100 E Fluorescence s 4 ty o RESULTS INTERPRETATION For each control and patient specimen calculate the concentration of HIV RNA usin
22. plasma using the Magno Sorb extraction kit Sacace K 2 16 1000 and RotorGene 6000 Target region pol gene TROUBLESHOOTING 1l Weak Ct gt 34 signal of the IC Fam channel retesting of the sample is required e The PCR was inhibited Make sure that you use a recommended RNA extraction method and follow the manufacturer s instructions 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 gt Make attention during the RNA extraction procedure 2 Weak Ct gt 40 signal on the Cy3 channel retesting of the sample is required 3 The correlation coefficient R is less than 0 9 retesting of all samples is required 4 The calculated concentrations of HIV Rec Posl and or HIV Rec Pos2 are different from given control concentrations reported in the Data Sheet retesting of all samples is required 5 Any signal on the Joe HEX Cy3 channel with Negative Control of extraction e Contamination during RNA extraction procedure All samples results are invalid gt
23. played threshold position and enter 30 into the Threshold Position field Click Recalculate Threshold Cycles Make certain that in the window Select Analysis Mode is selected PCR Baseline Subtracted Curve Fit iLycle Thicycler xl Servkes Help Services Help View Save Data PER Quantification PCR St d Curve Mek Curve N A ic Discrimination N A View Save Data PCR Quantification PCR Standard Curve Melt Curve N A Allelic Discrimination N A andar Allelic Data File Data 13 Nov 06 1634 0pd FAM 4 Data File Data 03 Feb 05 1758 0pd JOE 530 P select Wells Select a Reporter Select Wells ne Sere FAM 490 Select analysis mode PCR Base Line Subtracted Curve Fi r Threshold Cycle Calculation L EF KIE Reports Recalculate Threshold Cycles Select analysis mode Por Base Line Subtracted Curve Fit zj Threshold Cycle Calculation Baseline Cycles L 208 530 LI Reports Baseline Cycles Auto Calculated C User Defined Threshold Position C Auto Calculated f Auto Calculated Save for X axis Allelic Analysis Threshold Position 30 0 C User Defined C Auto Calculated User Defined Log View Save for X axis Allelic Analysis Pi Save for Y axis Allelic Analysis Workshop l so User Defined Log View _Save for Y axis Allelic Analysis Save for Y axis Allelic Analysis lt Threshold
24. s and select None as passive reference dye as shown in the following pictures Select the dye to 5 Double click on each standard well a new small window will appear and enter both for HCV HBV or HIV and Internal control the corresponding concentration in the Quantity field as indicated in the Data Card 6 Click on fi nes insert Reaction volume 25 ul HIV Real TM Ouant Number ofCycles 5 5 Number of Cycles 40 50 C 30 00 95 C 15 00 95 C 0 20 4 Spi Sep 1 55 C 0 40 40 Legend 72 C 0 30 Data Collection On Data Collection Off 7 Click to begin PCR reaction Analysis Settings 8 After the run is complete select all samples and standards and click 9 Deselect Use default settings Automatic threshold and automatic baseline suggested values are Ct setting for HIV Ct setting for Internal Control CT Settings to Use Use Default Settings Cr Settings to Use Use Default Settings Automatic Threshold C Automatic Threshold Threshold 30 000 Threshold 35 000 Automatic Baseline Automatic Baseline Baseline Start Cycle 3 End Cycle 15 4 Baseline Start Cycle 3 End Cycle 15 5 for StepOne instrument recommended Threshold values are Joe HIV 1 000 Fam IC 2 000 NOTE please slightly adapt suggested values according to your result curves acace
25. the option New Experiment Advanced Setup insert experiment details Select your Instrument Type 7500 7500 fast StepOne StepOne Plus Quantitation Standard curve Taqman Reagents Standard ramp speed DO NOT select Fast RampSpeed Which instrument are you using to run the experiment x Which reagents do you want to use to detect the target sequence Set up run and analyze an experiment using a 4 or 5 color 96 well system The PCR reacticns cottain primers des gned to amialify the targat sequence and a TaqMan probe t Which ramp speed do you want to use in the instrument run For optimal results with the standard ramp speed Applied Biosystems reconmerds using standart What type of experiment do you want to set up Use standards to determine the absolute quantity oftargel nucleic acid sequence in samples Note when using StepOne software be sure to click New Experiment Advanced Setup File Edit Instrument Analysis Advanced Setup n Open Design Wizard 9 Setup anew From Template 2 Click EM add two targets one for HIV Reporter Joe and the other one for Internal Control Reporter Fam select Quencher None for both targets 3 Click Add new samples multiple times and enter name for all samples controls and standards 4 Click Assign target and Samples tab associate samples uU and standards EA with the two target
26. utions when performing the assay Clean and disinfect all spills of specimens or reagents using a disinfectant such as 0 5 sodium hypochlorite or other suitable disinfectant Follow by wiping down the surface with 70 ethanol Avoid contact of specimens and reagents with the skin eyes and mucous membranes If these solutions 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 Workflow in the laboratory must proceed in a uni directional manner beginning in the Extraction Area and moving to the Amplification Area Do not return samples equipment and reagents in the area where you performed previous step Personnel should be using proper anti contamination safeguards when moving between areas STORAGE INSTRUCTIONS Part N Controls must be stored at 2 8 C Part N 2 HIV Real TM Quant must be stored at 20 C The kit 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 HIV Real TM Quant Test 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 thaw
27. way during the child delivery or soon after the childbirth at breast feeding One of the most effective current methods of direct HIV detection is specific amplification of nucleic acids in vitro by polymerase chain reaction PCR This method has a lot of advantages e detection of virus DNA RNA allows reducing the length of the serological window e PCR is an indispensable approach for HIV diagnostics in children born from HIV infected mothers e determination of HIV RNA in the blood plasma viral load is an obligatory procedure to monitoring of the therapy effectiveness INTENDED USE kit HIV Real TM Quant is a Real Time test for the quantitative detection of Human Immunodeficiency Virus HIV and simultaneous detection of a HIV specific Internal Control IC by dual color detection PRINCIPLE OF ASSAY kit HIV Real TM Quant is a Real Time test for the quantitative detection of Human Immunodeficiency Virus in human plasma HIV RNA is extracted from plasma amplified using real time amplification and detected using fluorescent reporter dye probes specific for HIV or HIV IC Internal Control IC serves as an amplification control for each individually processed specimen and to identify possible inhibition IC is detected in a channel other than the HIV RNA HIV Rec IC is a quantitative Internal Control concentration reported in Data Card and represents recombinant RNA containing structure which carried through all steps of analysis from nucl
Download Pdf Manuals
Related Search