HIV PCR detection Kit USER MANUAL
Contents
1. DNA Technology Research amp Production LLC declares that the above mentioned products meet the provision of the Council Directive 98 79 EC for In Vitro Diagnostic Medical Devices The quality control procedures performed in accordance with ISO ISO 9001 2008 and ISO 13485 2003 14 15 KEY TO SYMBOLS lt s O d lt m AJ MELES Authorized representative in EU Caution Consult instructions for use Date of manufacture Expiration date In vitro diagnostic medical device Batch code Version R3 P609 23 9EU F1 P609 52 1bEU R3 P609 S3 9EU F1 P609 21 1EU R3 P609 24 9EU F1 P609 51 1EU Upper limit of temperature Manufacturer ONTROL Negative control Positive control Cataloque number Sufficient for g jE ia n Temperature limitation 311 12 03 2014 ER lt 15 162. All components of the HIV PCR detection Kit except PCR mix and C must be stored at minus 20 C over the storage period The PCR buffer and mineral oil can be stored at at 2 8 C The PCR mix C and PREP NA DNA RNA Extraction Kit must be stored at 2 8 C over the storage period 13 Transportation can be held by all types of roofed transport with adherence to above mentioned temperature requirements An expired HIV PCR detection Kit must not be used We strongly recommend following the instructions to get robust and reliable results The conformity of the HIV PCR detection Kit to the prescribed technical requirements is subject to compliance of storage carriage and handling conditions recommended by manufacturer Contact our customer service by quality issues of the HIV PCR detection Kit 115587 Moscow Varshavskoye sh 125g building 6 DNA Technology LLC Phone Fax 7 495 9804555 e mail help dna technology ru www dna technology ru 13 SPECIFICATIONS a Analytical specificity the HIV PCR detection Kit allows detection of HIV 1 The samples containing HIV 1 will be defined as positive The samples not containing HIV 1 will be defined as negative b Sensitivity not less than 200 copies of HIV DNA per 1 mL of blood plasma c Diagnostic sensivity 99 5 d Diagnostic specificity 100 A The claimed specifications are guaranteed when DNA extraction is performed with PREP NA DNA RNA Extraction Kit 14 QUALITY CONTROL
3. be performed in each of two variants real time and end point fluorescent detection FLASH technology The HIV REAL TIME PCR Detection Kit is based on fluorescent modification of the PCR method The PCR mix contains target specific probes bearing reporter and quencher molecules Once hybridized to a target sequence the probes become activated As a result of activation fluorescence increases proportionally to target sequence amplification The intensity of fluorescence is measured at every cycle of reaction with a Real time PCR thermal cycler data collection unit and analyzed with the software provided The HIV FLASH PCR Detection Kit is based on the same principle but the fluorescence is measured only once after reaction The automatic analysis for HIV REAL TIME PCR Detection Kit is available on DNA Technology made instruments DT ite and DTprime Thermal Cyclers see the catalogue at www dna technology ru en to see available supply options and for HIV FLASH PCR Detection Kit on Gene or Gene 4 Fluorescence Readers REF cen E EU O GENE4 EU The HIV REAL TIME PCR Detection Kit is also approved for use with iQ Bio Rad Laboratories and Rotor Gene Qiagen real time thermal cyclers The HIV FLASH PCR Detection Kit is also approved for use with Ala1 4 fluorescence reader BioSan 3 CONTENT The Kit consists of three parts namely PREP NA DNA RNA Extraction Kit Reverse Transcription Kit and HIV PCR detection Kit either Real time or FLA
4. tubes for 15 min at 65 C spin down the drops at 13000 rpm for 30 s at room temperature 18 25 C Add 400 ul of the precipitation buffer into all tubes Close the tubes and mix them for 3 5 s twice Spin the tubes at 13000 rpm for 15 min at room temperature 18 25 C Remove the supernatant avoiding contact of the pipette tip with the precipitate Use new tip for each sample Add 500 ul of the washout solution Ne1 to the precipitate and shake the tube thoroughly 6 15 6 16 6 17 6 18 6 19 6 20 6 21 6 22 6 23 Spin the tubes at 13000 rpm for 5 min at room temperature 18 25 C Remove the supernatant avoiding contact of the pipette tip with the precipitate Use new tip for each sample Add 300 ul of the washout solution N92 to the precipitate and shake the tube thoroughly Spin the tubes at 13000 rpm for 5 min at room temperature 18 25 C Remove the supernatant avoiding contact of the pipette tip with the precipitate Use new tip for each sample Open the tubes and dry the precipitate at 65 C for 5 min Add 16 5 uL of the elution buffer to the precipitate Spin down the drops for 3 5 s Incubate the tubes for 10 min at 65 C Spin down the drops at 13000 rpm for 30 s 7 REVERSE TRANSCRIPTION PROTOCOL 7 1 7 2 Thaw content of RT buffer and RT mix tubes from Reverse Transcription Reagent Set at room temperature then vortex thoroughly and spin down drops by centrifuging at 1000 3000 RPM
5. For professional use only HIV PCR detection Kit PREP NA DNA RNA Extraction Kit included USER MANUAL DNA Technology Research amp Production LLC Russia 142281 Moscow Region Protvino 20 Zheleznodorozhnaya Street Phone fax 7 495 980 45 55 7 4967 31 06 70 E mail protvino dna technology ru mail dna technology ru http www dna technology ru R3 P609 23 9EU F1 P609 52 1bEU R3 P609 S3 9EU F1 P609 21 1EU 311 12 03 2014 REF R3 P609 24 9EU VER F1 P609 51 1EU Table of contents 10 11 12 13 14 15 INTENDED USE METHOD CONTENT REAGENTS AND EQUIPMENT REQUIRED BUT NOT PROVIDED WARNINGS AND PRECAUTIONS RNA EXTRACTION PROTOCOL REVERSE TRANSCRIPTION PROTOCOL PCR PROTOCOL CONTROLS DATA ANALYSIS TROUBLESHOOTING STORAGE AND HANDLING REQUIREMENTS SPECIFICATIONS QUALITY CONTROL KEY TO SYMBOLS 12 12 13 13 14 14 15 1 INTENDED USE The HIV PCR detection Kit is intended for research and diagnostic applications The HIV PCR detection Kit is an in vitro Nucleic Acid Test NAT based pathogen detection product The HIV PCR detection Kit is designed to detect Human Immunodeficiency Virus type 1 HIV 1 nucleic acids in human blood plasma The HIV PCR detection Kit can be used in clinical practice for HIV diagnostics 2 METHOD The implemented method is based on viral RNA reverse transcription followed by PCR amplification of the obtained cDNA The detection can
6. SH variant each is providing the crucial step of the assay Table 1 PREP NA DNA RNA Extraction Kit ne buffer Colorless liquid L 1 25 mL i SE erin 4 tubes Dissolvi ff iqui issolving buffer Colorless liquid each tube 3 mL 1 5 mL in each tube Internal control RNA IC Colorless liquid Table 2 Reverse Transcription Kit RT buffer Colorless liquid 200 uL Negative control C Colorless liquid 2 tubes LU 3 sum Colorless liquid 100 uL Table 3 HIV PCR detection Kit 96 or 100 separate or stripped tubes of 0 2 or 0 5 mL TECHNO Taq polymerase Colorless viscous liquid 50 uL PCR buffer Colorless liquid Se ams 2 tubes each tube Positive control C Colorless liquid 150 uL Mineral oil not supplied 2 mL 1 mL in each in Kit for Rotor Gene tube Applicable to FLASH PCR kits Colorless liquid and 1 92 mL or 2 0 mL white waxy fractions 0 02 mL per tube Paraffin sealed PCR mix Colorless viscous liquid The approximate total time needed to perform the assay is 5 hours The PREP NA DNA RNA Extraction Kit is sufficient for extraction of 100 samples The HIV PCR detection Kit sufficient to test 96 100 samples including IC negative and positive control samples 4 REAGENTS AND EQUIPMENT REQUIRED BUT NOT PROVIDED 4 1 Specimen collection The blood samples should be collected in 2 or 4 mL Vacuette type tubes with EDTA in 2 0 mg mL final concentration The sodium citrate anticoagulant is
7. also applicable N The use of heparin anticoagulant is not allowed 4 2 RNA extraction and PCR Vortex mixer 0 2 0 5 and 1 5 mL tubes PCR tube rack for 0 2 0 5 and 1 5 mL tubes Single channel pipettes volume range 2 20 ul 20 200 ul 200 1000 ul RNase and DNase free filtered pipette tips volume range 20 ul 200 ul 1000 ul Powder free surgical gloves Disinfectant solution Container for used pipette tips High speed centrifuge 13000 rpm Thermostat temperature range 50 95 C Real time PCR thermal cycler for HIV REAL TIME PCR Detection Kit Tercyc Conventional PCR Thermal Cycler REF a cu or equivalent for HIV FLASH PCR Detection Kit Gene or Gene 4 Fluorescence Reader REF cene cu O GENE4 EU or Alai 4 fluorescence reader or equivalent for HIV FLASH PCR Detection Kit 5 WARNINGS AND PRECAUTIONS The laboratory makeup should comply the requirements regulating work with microorganisms of I IV classes of pathogenicity Handle and dispose all biological samples reagents and materials used to carry out the assay as if they were able to transmit infective agents Avoid direct contact with the biological samples reagents and materials used to carry out the assay Any material coming in contact with the biological samples must be treated for at least 30 minutes with disinfecting solution or autoclaved for 1 hour at 1219C before disposal Molecular biology procedures such as nucleic acids extraction re
8. for 3 5 sec Prepare RT mix by mixing together RT Buffer RT sum and reverse transcriptase in separate plastic tube e 2 0 x N 1 ul of the RT buffer 1 0 x N 1 ul of the RT sum e 0 5 x N 1 ul of the reverse transcriptase where N 1 is the number of samples being analyzed considering C N and one extra sample CAUTION Reverse transcriptase should be kept out of freezer chamber for as short time as possible 7 3 7 4 7 5 7 6 7 7 Vortex RT mix and spin down drops by centrifuging at 1000 3000 RPM for 3 5 sec Transfer tube with RT mix to NA extraction room Add 3 5 ul of the RT mix to each tube with isolated RNA sample and to C tube Pipette 5 7 times to mix the content of the tube Place tubes in thermostat and incubate at 400C for 30 min then heat up to 950C and leave for 5 min Spin the tubes at 13000 RPM for 30 sec to collect the drops The cDNA preparation is ready for PCR Note The storage of the cDNA preparation is allowed at minus 20 C for not longer than one month 8 PCR PROTOCOL 8 1 Mark tubes with PCR mix for each test sample negative control C positive control C and two tubes for background buffer applicable to FLASH PCR kits For example if you need to test 10 samples mark 12 tubes 10 for each sample 1 for C 1 for C For FLASH PCR kit mark 14 tubes 10 for each sample 1 for C 1 for C and 2 for background buffer N Mark only the caps of the tubes
9. ime Setpoint 2C d 25 Acquisition dynamicwf tmo program ENMNM D M OO OP OO LLL Lee NENNEN Ne D 1 3 1 99 8 Wemme ENENNM o0 0 m L 1 12 09320 940 Lo 2 0030 S80 J Lo 3 000 580 RealTime Lo 4 020 3 amp 0 J 5 l 3100 J Storage Table 8 The PCR program for Rotor Gene Thermal Cyclers Cycling Hold Time Cycle Repeats Cycling 2 EO B58 C a ae 25 sec Take the measurement Table 9 Detection channels Specific product 11 9 CONTROLS Table 11 Control Specific signal is Specific signal is Interpretation present absent C PCR lt extraction PCR and DNA E x wag RNA IC O a Valid extraction S a a Invalid OO a E ee a PCR and DNA The sample is considered positive if the signal for specific DNA is present The signal for IC could be absent in samples with high concentration of specific DNA due to competitive priming The sample is considered negative if the signal for specific DNA is absent and for RNA IC is present If the signal for C is present whole tests of current batch considered false Decontamination required 10 DATA ANALYSIS In case of using DNA Technology made Real Time PCR Thermal Cyclers or Fluorescence Readers the analysis performed automatically In all other cases the a
10. into corresponding tube Avoid paraffin layer break Do not add DNA into the C C and background applicable to FLASH PCR kits tubes Avoid paraffin layer break A Open the tube add DNA sample then close the tube before proceeding to the next DNA sample to prevent contamination Use filter tips 8 10 Add 5 0 ul of C which has passed NA isolation stage and reverse transcription reaction into C and background applicable to FLASH PCR kits tubes Add C into corresponding tube Avoid paraffin layer break 8 11 Spin tubes briefly 1 3 sec 8 12 Setthe tubes to the Thermal Cycler 8 13 Launch the Thermal Cycler software and run PCR according to instructions supplied with device considering 35 ul reaction mix volume See tables 4 8 to refer the cycling program and table 9 to refer the detection channels applicable to Real Time PCR kits Using Thercyc cycler you need to choose Rapid active regulation regulation algorithm Amplification programs correspondence with reagent kits is shown in Table 4 Table 4 The PCR program for Tercyc Conventional PCR Thermal Cycler For thermal cyclers with active regulation Number of cycles Time Optical measurement LR ee Number of Type of the Storage Storage Sen PCR Melt Data p Acquisition 94 0 00 25 58 0 00 15 at 0 i int 2C 94 0 0 0 10 Table 7 The PCR program for iCycler iQ thermal cyclers Bio Rad Laboratories PCR Melt Dat Cycle Repeats Step Dwell t
11. nalysis is based on the presence or absence of specific signal The controls should be also considered to exclude false positive and false negative results see p 7 of the current manual The cutoff Ct values for Rotor Gene thermal cycler are 40 specific product and 33 C The result characterized by Ct above this value should be considered doubtful and the whole assay should be repeated The analysis performed automatically After completion of the run the device will build standard curve define the concentration of viral DNA and form the report The PCR efficiency should be in 90 100 range The interpretation should be performed in accordance with table 12 12 Table 12 HIV FLASH PCR Test samples detection Kit HIV REAL TIME PCR detection Kit Interpretation Tem Cp not specified l for iQ N A T uncertain Cp not specified oem uncertain for iQ N A C N i Cp is specified Positive C d Cp not specified Cp 29 34 for iQ5 Ct 29 EMEN in 11 TROUBLESHOOTING Table 13 ific signal i Operation error Repeat whole test PCR inhibition C Violation of storage and Dispose current batch handling requirements Dispose current batch C Contamination Perform decontamination procedures RNA IC OUT w PCR inhibition Repeat whole test If you face to any undescribed issues contact our representative 12 STORAGE AND HANDLING REQUIREMENTS Expiry date 9 month from the date of production
12. t exceed 6 hours The transportation and storage temperature from collecting the sample till analysis should be in 2 8 C range 6 1 6 2 To obtain the plasma spin the tubes with blood at 3000 rpm for 20 min at room temperature 18 25 C Take the upper fraction plasma with an automatic sampler and put it into the new 1 5 mL tube The blood plasma can be stored at minus20 C for 3 months N The lysis buffer can contain the precipitate Dissolve it at 65 C for 10 min prior to use N At this step of assay use only RNase and DNase free pipette tips A To rise the reliability of the results it is advised to perform the extraction in duplicates 6 3 6 4 6 5 6 6 Mark the required number of 1 5 mL tubes by the following scheme for each test sample and for negative control C For example if you need to test 10 samples mark 11 tubes 10 for the samples 1 for C Add 10 uL of the premixed internal control RNA IC in each tube Add 300 ul of the lysis buffer avoiding contact of the pipette tip with an edge of the tube Close the tubes N Open the tube add sample then close the tube before proceeding to the next DNA sample to prevent contamination 6 7 6 8 6 9 6 10 6 11 6 12 6 13 6 14 Add 100 uL of the blood plasma sample into the marked tubes Do not add samples to the C tube Add 100 uL of the C into corresponding tube Close the tubes and mix them for 3 5 s twice Incubate the
13. verse transcription amplification and detection require qualified staff to avoid the risk of erroneous results especially due to the degradation of nucleic acids contained in the samples or sample contamination by amplification products All oligonucleotide components are produced by artificial synthesis technology according to internal quality control protocol and do not contain blood or products of blood processing Positive control is produced by artificial DNA synthesis technology Positive control does not include parts of infectious agents All the liquid solutions are designed for single use and can not be used more than once in amplification reactions Plastic tubes do not contain phthalates Do not breathe gas fumes vapour spray produced by the components of the kit Do not eat drink components of the kit Avoid contact with eyes Do not use the kit after the expiry date provided Only use the reagents provided in the kit and those recommended by manufacturer Do not mix reagents from different batches Do not use reagents from third party manufacturers kits Significant health effects are NOT anticipated from routine use of this kit when adhering to the instructions listed in the current manual 6 RNA EXTRACTION PROTOCOL The HIV PCR detection Kit is designed to detect DNA extracted from blood plasma Shake the tube containing blood sample thoroughly to mix the blood and anticoagulant N The overall storage of the sample should no
14. when using Rotor Gene Thermal Cycler 8 2 Thaw PCR buffer at the room temperature 8 3 Mix the PCR buffer and TECHNO Taq polymerase thoroughly 3 5 sec then spin briefly 1 3 sec at room temperature 18 25 C N Hold TECHNO Taq polimerase at room temperature as short time as possible The overheating is detrimental to its performance 8 4 Prepare the mixture of PCR buffer and TECHNO Taq polymerase Taq polymerase solution Add into the one tube e 10 x N 1 uL of PCR buffer e 0 5 x N 1 uL of TECHNO Taq polymerase N number of the marked tubes including C C background tubes For example if you need to test 10 samples 12 marked tubes prepare mixture of PCR buffer and TECHNO Taq polymerase for 13 1241 tubes 130 uL PCR buffer 6 5 uL TECHNO Taq polymerase 8 5 Vortex the tube with Taq polymerase solution for 3 5 seconds and spin down the drops for 1 3 seconds at room temperature 18 25 C The maximum storage time for Taq polymerase solution is 1 hour 8 6 Add 10 ul of Taq polymerase solution into each tube except background tubes Add 10 ul of background buffer into corresponding tubes applicable to FLASH PCR kits Avoid paraffin layer break 8 7 Add one drop 20 ul of mineral oil into each tube not applicable to kits approved for use with Rotor Gene thermal cycler Close tubes tightly 8 8 Vortex the tubes with samples for 3 5 seconds and spin down the drops for 1 3 seconds 8 9 Add 5 0 ul of DNA sample
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