HCV Quantitative Real-Time PCR Kit USER MANUAL
Contents
1. 58 0 L3 Ll l 10 J Storage 10 Table 7 The PCR program for Rotor Gene Thermal Cyclers Take the measurement Table 8 Detection channels Te Specific product DTprime DTlite and 105 9 CONTROLS Table 9 Result thecontratied OOOO O ee un Contro step Specific signal is Specific signal is nterpretation present absent BER PCR and RNA extraction o 7 Vaid PCR and RNA 2 kelk IC Valid extraction Invalid 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 11 10 DATA ANALYSIS 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 10 Table 10 Detection channel Fam Green Interpretation l Hex Yellow Cp Ct copies ml Test samples Positive with specified viral load 7 5x10 1 0x10 Not considered copies ml Positive with notification Less than Less than 7 5x107 Not cons2. 3 seconds Add 5 0 uL of DNA sample into corresponding tube Avoid paraffin layer break N 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 8 11 8 12 Add 5 0 uL of C C ST1 and ST2 into corresponding tubes Avoid paraffin layer break Spin tubes briefly 1 3 sec Set the tubes to Real Time PCR Termal Cycler 8 13 Launch the Thermal Cycler software and run PCR according to instructions supplied with device See table 4 7 to refer the cycling program an table 8 to refer the detection channels Table 4 The PCR program for DT ite and DT prime Thermal Cyclers Optical measurement Number of Type of the step 1 Ode ae EI poe Holing Table 5 The PCR program for iCycler iQ5 thermal cyclers with dynamic factor PCR Melt Dat Cycle Repeats Step Dwell time Setpoint 9C gt ix Acquisition dynamicwf tmo program EXEM i EE g s f ve y o 1 a E D 13 39 8 WeWme NENNEN 5 hA JMhA Lh L0 1 P 1o 94 Lo min 62 RealTime 5 e l 10 J strae Table 6 The PCR program for iCycler iQ5 thermal cyclers with persistent well factor PCR Melt Dat Cycle Repeats Step Dwell time Setpoint 9C E ns Acquisition Oo c 1L iL OT LU min 494 DJ L2 5 Jj j P qo p jun 1 10s 94
3. A EXTRACTION PROTOCOL The HCV Quantitative Real Time PCR 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 4 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 N To rise the reliability of the results it is advised to perform the extraction in duplicates 6 3 Mark the required number of 1 5 ml tubes by the following scheme 2 tubes for each sample to be tested 1 tube for the negative control C 3 tubes for ST1 3 tubes for ST2 For example if you need to test 10 samples mark 27 tubes 20 for the samples 1 for C 3 for ST1 3 for ST2 6 4 Add 10 uL of the premixed RNA IC in each tube except ST1 and ST2 6 5 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
4. For professional use only HCV Quantitative Real Time PCR 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 Q4 P603 23 9EU Q4 P603 S3 9EU Q4 P603 24 9EU ER 252 11 04 13 lt 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 11 12 13 13 13 14 15 1 INTENDED USE The HCV Quantitative Real Time PCR Kit is intended for research and diagnostic applications as well as for evaluation of the therapy efficacy The HCV Quantitative Real Time PCR Kit is an in vitro Nucleic Acid Test NAT based pathogen detection and quantification product The HCV Quantitative Real Time PCR Kit is designed to detect and quantitate Hepatitis C Virus HCV nucleic acids in human blood plasma samples with an aid of Quantitative Real Time Polymerase Chain Reaction qPCR method The HCV Quantitative Real Time PCR Kit can be used in clini
5. alysis The quantitation of the target RNA is performed with an aid of Standards ST with known concentration of artificially synthesized target DNA The Kit supplied with STs of the two concentrations 1x10 ST1 and 3x10 copies ml ST2 The STs are used to build the standard curve which is necessary to quantitate the RNA in the sample 3 CONTENT Table 1 PREP NA DNA RNA Extraction Kit ne buffer Colorless liquid 5 ml 1 25 ml in joue Elution buffer Colorless liquid each tube 3 ml 1 5 ml in each tube Internal control RNA IC Colorless liquid Table 2 Standards 1 5 ml 0 3 ml in each tube Negative control C Colorless liquid 2 tubes ST1 1x10 copies ml Colorless liquid 5 tubes 1 5 ml 0 3 ml in each tube aunes ST2 3x10 copies ml Colorless liquid Table 3 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 4 HCV Quantitative Real Time PCR Kit Composition of colorless a 1 92 ml 0 02 uL 96 separate 1x96 or Paraffin sealed PCR mix liquid and white waxy PeriHbe saonediad MUSS fractions TECHNO Taq polymerase Colorless viscous liquid 50 uL PCR buffer Colorless liquid pn in 2 tubes each tube Positive control C Colorless liquid 150 uL Mineral oil not supplied in 2 ml 1 ml in each ETO Kit for Rotor Gene tube Colorless viscous liquid The a
6. bes 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 uL 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 ST1 ST2 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 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 Place tubes in thermostat and incubate at 40 C for 30 min than incubate at 95 C for 5min 7 6 Spin down condensate by centrifuging at 13000 RPM for 30 sec cDNA preparation is ready for carrying out PCR Note cDNA storage at 20 C for not longer than one month is tolerated 8 PCR PROTOCOL 8 1 Mark tubes wi
7. cal practice for HCV diagnostics 2 METHOD The implemented PCR method is based on amplification of a target DNA sequence The HCV Quantitative Real Time PCR Kit is based on RNA reverse transcription process and consequential cDNA fragments amplification with polymerase chain reaction PCR method The amplification process lies in repeated cycles thermal DNA denaturing primer annealing with complementary sequences and further polynucleotide chains completion by Tag polymerase An internal control sample corresponding to a stabilized RNA fragment is added to a sample being examined at the stage of nucleic acids isolation and intended for estimation of all the examination stages efficacy The HCV Quantitative Real Time PCR Kit DNA probes each of which contains a fluorescent label and fluorescence quencher are included in PCR mix In case of specific cDNA product formation a probe gets destroyed and that leads to fluorescence level growth registered by special appliances DNA probes used for sought nucleic acid NA and internal control IC PCR products detection are labeled with FAM and HEX fluorescent probes accordingly That allows separate Hepatitis C virus cDNA and internal control sample PCR results registration For PCR products analysis detecting PCR cyclers should be used For reaction sensitivity and specificity enhancement application of hot start ensured by a two layer reaction mix divided with a paraffin streak preparation met
8. hod is provided Mixing the layers and turning them into PCR mix occurs only with paraffin melting That eliminates non specific primer annealing on target DNA upon tube preheating The HCV Quantitative Real Time PCR Kit is based on real time detection of the target DNA sequence Real time PCR technology is based on measurement of the fluorescence at every cycle of reaction The PCR mix contains target specific hydrolyzing probes bearing reporter and quencher molecules Once hybridized to a target sequence the probe become activated As a result of activation fluorescence increases proportionally to target sequence amplification The intensity of fluorescence is detected with a real time PCR thermal cycler data collection unit and analyzed with the software provided The assay includes following steps RNA extraction On this step the internal control sample IC is added to the samples It is needed for test quality assurance RNA reverse transcription process to obtain cDNA for PCR Real time PCR amplification The Kit has passed validation on DNA Technology made instruments and software en O DTPRIME5M1 EU O DTLITEAS1 EU O DTLITE5S1 EU The HCV Quantitative Real Time PCR Kits BELL sins ania Q4 P603 S3 9EU and Q4 P603 24 9EU are also approved for use with iQ5 Bio Rad Laboratories and Rotor Gene Qiagen thermal cyclers respectively The Kit can be supplied in either separate 1x96 or stripped 8x12 tubes Quantitative an
9. idered m 750 copies ml no specified value Positive with notification More More than 1 0x10 Not considered than 1 0x10 copies ml no specified value PN Specified for DT ite DTprime Not fied N A Negative ot specified N A Cp Ct 29 34 gativ Not specified N A dad i C 2 5x10 8 0x10 Not considered Positive with specified viral load copies ml Not specified Cp Ct 29 34 Negative If the concentration of the C falls out the 2 5x10 8 0x10 range the test should be repeated 12 11 TROUBLESHOOTING Table 11 Operation error Repeat whole test PCR inhibition Violation of storage and Dispose current batch handling requirements Dispose current batch Contamination Perform decontamination procedures 12 STORAGE AND HANDLING REQUIREMENTS Expiry date 6 month from the date of production All components of the HCV Quantitative Real Time PCR Kit except PCR mix ST1 ST2 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 ST1 ST2 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 An expired HCV Quantitative Real Time PCR Kit must not be used We strongly recommend following the instructions to get robust and reliable results The conformity
10. itive control Cataloque number Sufficient for Temperature limitation Upper limit of temperature 15 16
11. of the HCV Quantitative Real Time PCR 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 Quantitative Real Time PCR 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 Quantitative Real Time PCR Kit allows detection next HCV genotypes 1a 1b 2a 2b 2c 2i 3 4 5a 6 The samples containing HCV will be 13 defined as positive and characterized quantitatively The samples not containing HCV will be defined as negative b Linear range 7 5x10 1x10 copies ml c Variation coefficient less than 7 d Sensitivity not less than 200 copies of HBV DNA per 1 ml of blood plasma A The claimed specifications are guaranteed when DNA 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 lt O 8 of lt m AJ 15 KEY TO SYMBOLS Caution Consult instructions for use Date of manufacture Expiration date In vitro diagnostic medical device Batch code Version 3BE PE Manufacturer Negative control Pos
12. pproximate 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 HCV Quantitative Real Time PCR Kit sufficient to test 44 samples in duplicates 4 REAGENTS AND EQUIPMENT REQUIRED BUT NOT PROVIDED 4 1 Specimen collection The whole blood samples shoud 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 Vacuum pump with collector to remove the supernatants 1 5 ml tubes PCR tube rack for 0 2 and 1 5 ml tubes Single channel pipettes volume range 0 5 10 LL 5 40 uL 40 200 LL 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 40 95 C Refrigerator Real time PCR thermal cycler 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 ma
13. sample to prevent contamination 6 6 Add 100 uL of the blood plasma sample into the marked tubes Do not add samples to the C and ST tubes 6 7 Add 100 uL of the C ST1 ST2 into corresponding tubes 6 8 Close the tubes and mix them for 3 5 s twice 6 9 6 10 6 11 6 12 6 13 6 14 6 15 6 16 6 17 6 18 6 19 6 20 6 21 6 22 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 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 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 No2 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 dissolving buffer to the precipitate Spin down the drops for 3 5 s Incubate the tu
14. terial 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 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 RN
15. th PCR mix for each test sample negative control C positive control C and three tubes for each of the Standards ST1 and ST2 For example if you need to test 10 samples mark 28 tubes 20 for each sample in duplicate 1 for C 1 for C 3 for ST1 and 3 for ST2 N Mark only the caps of the tubes when using Rotor Gene Thermal Cycler 8 2 8 3 Thaw PCR buffer at the room temperature 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 8 5 8 6 8 7 8 8 8 9 Prepare the mixture of PCR buffer and TECHNO Taq polymerase TECHNO Taq polymerase solution Add into the one tube 10 x N 1 uL of PCR buffer 0 5 x N 1 uL of TECHNO Taq polymerase N number of the marked tubes including C C ST1 and ST2 Vortex the tube with TECHNO 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 Add 10 uL of TECHNO Taq polymerase solution into each tube Avoid paraffin layer break Add 20 uL of mineral oil into each tube Avoid paraffin layer break skip this step when using Q4 P603 24 9EU for Rotor Gene Close the tubes Vortex the tubes with samples for 3 5 seconds and spin down the drops for 1
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