HCV PCR detection Kit USER MANUAL
Contents
1. dissolving 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 The RNA preparation is ready RNA should be use immediately for reverse transcription reaction RNA sample shouldn t be stored 7 CARRYING OUT REVERSE TRANSCRIPTION REACTION 7 1 7 2 Thaw content of RT Buffer and RT HAV HCV HDV HGV HIV dNTP tubes from Reverse Transcription Reagent Set at room temperature then vortex thoroughly and spin down drops by centrifuging at 1000 3000 RPM for 3 5 sec Prepare the mixture of RT Buffer RT HAV HCV HDV HGV HIV dNTP and reverse trancriptase RT mix Add into the one plastic tube 2 0 x N 1 RT Buffer 1 0 x N 1 ul RT HAV HCV HDV HGV HIV dNTP 0 5 x N 1 ul reverse transcriptase where N 1 the number of samples being analyzed considering C N and one extra sample N 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 obtained and spin down drops by centrifuging at 1000 3000 RPM for 3 5 sec Add 3 5 ul RT mix to each tube with isolated RNA sample and to C tube Vortex tubes and sediment drops with centrifuging at 1000 3000 RPM for 3 5 sec Place tubes in thermostat and incubate at 400C for 30 min than incubate at 950C for 5 min Spin down condensate by centrifuging at 13000 RPM for 30 sec CDNA preparati2. tube Close the tubes N Open the tube add sample then close the tube before proceeding to the next sample to prevent contamination 6 7 Add 100 uL of the blood plasma sample into the marked tubes Do not add samples to the C tube 6 8 Add 100 uL of the C into corresponding tube 6 9 Close the tubes and mix them for 3 5 s twice 6 10 Incubate the tubes for 15 min at 65 C spin down the drops at 13000 rpm for 30 s at room temperature 18 25 C 6 11 A Add 400 of the precipitation buffer into all tubes Close the tubes and mix them for 3 5 s twice 6 12 Spin the tubes at 13000 rpm for 15 min at room temperature 18 25 6 13 Remove the supernatant avoiding contact of the pipette tip with the precipitate Use new tip for each sample 6 14 500 of the washout solution 1 to the precipitate and shake the tube thoroughly 6 15 Spin the tubes at 13000 rpm for 5 min at room temperature 18 25 6 16 Remove the supernatant avoiding contact of the pipette tip with the precipitate Use new tip for each sample 6 17 Add 300 ul of the washout solution Ne2 to the precipitate and shake the tube thoroughly 6 18 Spin the tubes at 13000 rpm for 5 min at room temperature 18 25 6 19 Remove the supernatant avoiding contact of the pipette tip with the precipitate Use new tip for each sample 6 20 Open the tubes and dry the precipitate at 65 C for 5 min 6 21 6 22 6 23 Add 25 uL of the
3. 80 bp Not considered Positive Positive Doom Negative 12 Table 13 Results with HCV Flash and Real time PCR detection Kits HCV FLASH PCR Test samples detection Kit HCV Real time PCR detection Kit Interpretation Hex Yellow Cp Ct is specified Not considered 29 Cp not specified co Ct 28 34 uncertain Cp not specified Th genns d uncertain for iQ N A C not specified 55 for iQ N A Cp Ct 29 34 Negative 11 TROUBLESHOOTING Table 14 Operation error Repeat whole test PCR inhibition Violation of storage and Dispose current batch handling requirements Dispose current batch Perform decontamination procedures If you face to any undescribed issues contact our representative Contamination 12 STORAGE AND HANDLING REQUIREMENTS Expiry date 9 month from the date of production All components of the HCV PCR detection Kit except PCR mix and C must be stored at 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 Transportation can be held by all types of roofed transport with adherence to above mentioned temperature requirements 13 An expired HCV PCR detection Kit must not be used We strongly recommend following the instructions to get robust and reliable results The conformity of the HCV P
4. CR 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 HCV 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 HCV PCR detection Kit allows detection next HCV genotypes 1a 1b 2a 2b 2c 21 3 4 5a 6 The samples containing HCV will be defined as positive The samples not containing HCV will be defined as negative b Sensitivity not less than 200 copies of HCV RNA per 1 ml of blood plasma Diagnostic sensivity 99 8 d Diagnostic specificity 100 A The claimed specifications are guaranteed when RNA extraction is performed with PREP NA DNA RNA Extraction Kit 14 QUALITY CONTROL DNA Technology Research amp Production LLC declares that the quality control procedures performed in accordance with ISO ISO 9001 2008 and ISO 13485 2003 14 15 KEY TO SYMBOLS Caution Consult instructions for use Date of manufacture Expiration date In vitro diagnostic medical device a B RE Batch code lt m AJ Version Manufacturer Negative control Positive control Cataloque number Sufficient for Temperature limitation Upper limit of temperature 15 16
5. For professional use only HCV 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 R1 P603 23 9EU F1 P603 52 1EU R1 P603 S3 9EU F1 P603 21 1EU 250 20 03 13 REF R1 P603 24 9EU E3 P603 50 1EU F1 P603 51 1bEU E3 P603 20 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 CARRYING OUT REVERSE TRANSCRIPTION REACTION 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 HCV PCR detection Kit is intended for research and diagnostic applications The HCV PCR detection Kit is an in vitro Nucleic Acid Test NAT based pathogen detection product The HCV PCR detection Kit is designed to detect Hepatitis C Virus HCV nucleic acids in human blood plasma The HCV PCR detection Kit can be used in clinical practice for HCV diagnostics 2 METHOD The implemented PCR method is based on amplification of a target cDNA sequence The detection can be performed in each o
6. able to Real Time PCR kits Using Thercyc cycler you need to choose Precision active regulation regulation algorithm Table 4 The PCR program for Tercyc Conventional PCR Thermal Cycler applicable to Conventional PCR kits For thermal cyclers with active regulation Number of ooo ooo 0 p swa Table 5 The PCR program for Tercyc Conventional PCR Thermal Cycler applicable to FLASH PCR kits For thermal cyclers with active regulation Number of cycles Temperature Time mn se 7 oo 195 t eee 2 94 5 5 64 5 67 5 O 00 lbP A 3 DDO eee 94 3 1 64 5 67 5 __ Table 6 The PCR program for DT ite and DTprime Thermal Cyclers Optical Type of the measurement 10 Table 7 The PCR program for iCycler iQ5 thermal cyclers with persistent well factor PCR Melt Dat 1 00 20 E 1 do o9 99 Table 8 The PCR program for iCycler iQ5 thermal cyclers with dynamic factor PCR Melt Dat Cycle Repeats Step Dwell time Setpoint 2C se ds Acquisition dynamicwf tmo program c ae _ 9 JM ee Lo et 90 j J __ 2 020 620 QJ RealTime 5 _ Ll 3100 J Storage Table 9 The PCR program for Rotor Gene Thermal Cyclers Cycling Hold Time Cycle Repeats Take the measurement Table 10 D
7. es 4 REAGENTS AND EQUIPMENT REQUIRED BUT NOT PROVIDED 4 1 Specimen collection The whole 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 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 0 5 10 uL 5 40 uL 40 200 uL 100 1000 LL RNase and DNase free filtered pipette tips volume range 20 LL 50 uL 200 uL 1000 LL Powder free surgical gloves Disinfectant solution Container for used pipette tips High speed centrifuge 13000 rpm Thermostat temperature range 50 65 C Physiological saline solution 0 996 NaCl Sterile Real time PCR thermal cycler for HCV Real Time PCR Detection Kit Tercyc Conventional PCR Thermal Cycler a cu or equivalent for HCV FLASH PCR Detection Kit and HCV Conventional PCR Detection Kit Gene or Gene 4 Fluorescence Reader BEP citri O GENE4 EU or Ala1 A fluorescence reader or equivalent for HCV FLASH PCR Detection Kit For electrophoretic detection AC power supply electrophoretic chamber transilluminator 1 0 L volumetric flask distilled water 1 0 mm diameter steel wire 5 WARNINGS AND PRECAUTIONS The laboratory makeup should comply the requirements regulating work with microorgan
8. etection channels 9 CONTROLS Table 11 Result The controlled RE TUO Control Specific signal is Specific signal is Interpretation present absent PCR BH t extraction Valid PCR and RNA E Valid extraction ee omo Invalid Lm PCRandRNA The sample is considered positive if the signal for specific cDNA is present The signal for IC could be absent in samples with high concentration of specific cDNA due to competitive priming The sample is considered negative if the signal for specific cDNA is absent and for 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 analysis 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 interpretation should be performed in accordance with tables 12 13 Table 12 Results with HCV Conventional PCR detection Kit Specific product 253 bp Internal control 4
9. f three variants real time HCV Real Time PCR Detection Kit endpoint HCV FLASH PCR Detection Kit and HCV Conventional PCR Detection Kit The HCV 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 HCV FLASH PCR Detection Kit is based on the same principle but the fluorescence is measured only once after reaction HCV Conventional PCR Detection Kit is developed for PCR result detection by electrophoresis in the agarose gel The automatic analysis available on DNA Technology made instruments DT ite or DTprime REAL TIME Thermal Cyclers for HCV Real Time PCR Detection Kit see the catalogue at www dna technology ru en to see available supply options and Gene or Gene 4 Fluorescence Readers cene cu O GENE4 EU for HCV FLASH PCR Detection Kit The HCV Real Time PCR Detection Kit is also approved for use with iQ Bio Rad Laboratories and Rotor Gene Qiagen real time thermal cyclers The HCV FLASH PCR Detection Kit is also approved for use with Ala1 4 fluorescence reader BioSan RNA extrac
10. isms 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 reverse 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 ki
11. olymerase 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 Add 10 uL of TECHNO 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 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 Vortex the tubes with samples for 3 5 seconds and spin down the drops for 1 3 seconds Add 5 0 uL of cDNA sample into corresponding tube Avoid paraffin layer break Do not add cDNA into the C C and background applicable to FLASH PCR kits tubes Avoid paraffin layer break N Open the tube add cDNA sample then close the tube before proceeding to the next sample to prevent contamination Use filter tips 8 10 8 11 8 12 Add 5 uL of C which passed whole RNA extraction procedure and reverse transcription into C and background applicable to FLASH PCR kits tubes Add C into corresponding tube Avoid paraffin layer break Spin tubes briefly 1 3 sec Set the tubes to the Thermal Cycler Launch the Thermal Cycler software and run PCR according to instructions supplied with device considering 35 ul reaction mix volume See tables 4 9 to refer the cycling program and table 10 to refer the detection channels applic
12. on is ready for carrying out PCR Note cDNA storage at 20 C for not longer than one month is tolerated The nucleic acid preparation is ready 8 PCR PROTOCOL 8 1 Mark tubes with PCR mix for each test sample negative control C positive control C Mark additionally two tubes for background buffer applicable to FLASH PCR kits For example if you need to test 10 samples mark 12 tubes 10 for samples 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 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 polymerase 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 TECHNO Taq polymerase solution Add into the one tube 10 x N71 uL of PCR buffer 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 8 6 8 7 8 8 8 9 Vortex the tube with TECHNO Taq p
13. t 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 HCV PCR detection Kit is designed to detect RNA extracted from whole blood Shake the tube containing blood sample thoroughly to mix the blood and anticoagulant N The overall storage of the sample should not exceed 6 hours The transportation and storage temperature from collecting the sample till analysis should be in 2 8 C range 6 1 To obtain the plasma spin the tubes with blood at 3000 rpm for 20 min at room temperature 18 25 C 6 2 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 20 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 6 3 Mark the required number of 1 5 ml tubes by the following scheme for each test sample and for negative control C 6 4 For example if you need to test 10 samples mark 11 tubes 10 for samples 1 for C 6 5 Add 10 uL of the premixed internal control RNA IC in each tube 6 6 Add 300 of the lysis buffer avoiding contact of the pipette tip with an edge of the
14. tion On this step the internal control sample RNA IC is added to the samples It is needed for test quality assurance 3 CONTENT Table 1 PREP NA DNA RNA Extraction Kit Lysis buffer MORE S 30 ml 1 vial liquid 1 25 mli h Dissolving buffer Colorless liquid e ix Negative control C Colorless liquid 3 ml 1 5 ml in each tube Table 2 Reverse RNA Transctription PCR Kit RT buffer Colorless liquid 200 uL RT HAV HCV HDV HGV HIV dNTP Colorless liquid 100 uL Reverse transcriptase Colorless liquid 50 uL Table 3 HCV PCR detection Kit Color coded composition of 96 or 100 separate or MA 1 92 ml or 2 0 ml Paraffin sealed PCR mix liquid and white waxy stripped tubes of 0 2 0 02 mL per tube fractions or 0 5 ml TECHNO Taq polymerase Colorless viscous liquid 50 uL 1 ml 0 5 ml in PCR buffer Colorless liquid e chitub Positive control C Colorless liquid 150 uL Mineral oil not supplied in 2 ml 1 ml in each Kit for Rotor Gene tube Colorless viscous liquid The approximate total time needed to perform the assay is 5 hours Upon customer s request optional supply of a reagent kit for DNA electrophoretic detection is possible including Electrophoresis mix 9 55 g and Agarose gel 5 plates The PREP NA DNA RNA Extraction Kit is sufficient for extraction of 100 samples The HCV PCR detection Kit sufficient to test 96 100 samples including negative positive and internal sampl
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