HBV Quantitative Real-Time PCR Kit USER MANUAL
Contents
1. 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 7 2 7 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 A Hold TECHNO Taq polymerase at room temperature as short time as possible The overheating is detrimental to its performance 1 4 7 5 7 6 7 7 7 8 7 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 TECHNO Tag 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 Q2 P602 24 9EU Close the tubes Vortex the tubes with samples for 3 5 seconds and spin down the drops for 1 3 seconds Add 5 0 uL of DNA sample into corresponding tube Avoid paraffin lay2. Close the tubes and mix them for 3 5 s twice 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 1 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 2 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 25 of the dissolving buffer to the precipitate Spin down the drops for 3 5 s Incubate the tubes for 10 min at 65 C 6 22 Spin down the drops at 13000 rpm for 30 s The nucleic acid preparation is ready The preparation can be stored at 20 C for 1 month or at 70 C for 1 year 7 PCRPROTOCOL 71 Mark tubes with PCR mix for each test sample negative control C positive control
3. 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 DNA EXTRACTION PROTOCOL The HBV Quantitative Real Time PCR Kit is designed to detect DNA 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 transportati
4. CR Kit allows detection of all known HBV subtypes The samples containing HBV will be defined as positive and characterized quantitatively The samples not containing HBV will be defined as negative 12 b Linear range 7 5x10 1x10 copies ml c Variation coefficient less than 796 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 13 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 13 14 KEY TO SYMBOLS lt m A aa Caution Consult instructions for use Date of manufacture Expiration date In vitro diagnostic medical device Batch code Version ONTROL V 1 f Manufacturer Negative control Positive control Cataloque number Sufficient for Temperature limitation Upper limit of temperature 14 15 16
5. D 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 DNA 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 50 65 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 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
6. For professional use only HBV 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 Q2 P602 23 9EU Q2 P602 S3 9EU VER 280 08 11 13 Q2 P602 24 9EU Table of contents 10 11 12 13 14 INTENDED USE METHOD CONTENT REAGENTS AND EQUIPMENT REQUIRED BUT NOT PROVIDED WARNINGS AND PRECAUTIONS DNA EXTRACTION PROTOCOL 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 HBV Quantitative Real Time PCR Kit is intended for research and diagnostic applications as well as for evaluation of the therapy efficacy The HBV Quantitative Real Time PCR Kit is an in vitro Nucleic Acid Test NAT based pathogen detection and quantification product The HBV Quantitative Real Time PCR Kit is designed to detect and quantitate Hepatitis B Virus HBV nucleic acids in human blood plasma samples with an aid of Quantitative Real Time Polymerase Chain Reaction qPCR method The HBV Quantitative Real Time PCR Kit can be used in clinical practice for HBV diagnostics 2 METHOD The implemented
7. PCR method is based on amplification of a target DNA sequence The HBV 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 DNA extraction On this step the internal control sample IC is added to the samples It is needed for test quality assurance Real time PCR amplification The Kit has passed validation on DNA Technology made instruments and software ec O DTPRIME5M1 EU O DTLITEAS1 EU O DTLITE5S1 EU The HBV Quantitative Real Time PCR Kits REF A Q2 P602 S3 9EU and Q2 P602 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 Bio cum Q2 P602 24 9EU and Q2 P602 S3 9EU respectively Quantitative analysis The quantitation of the target DNA is performed with an aid of Standards ST with known concentration of artificially synthesiz
8. e test should be repeated 11 10 TROUBLESHOOTING Table 11 Operation error Repeat whole test PCR inhibition Dispose current batch Violation of storage and handling requirements Dispose current batch Contamination Perform decontamination procedures 11 STORAGE AND HANDLING REQUIREMENTS Expiry date 6 month from the date of production All components of the HBV 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 HBV Quantitative Real Time PCR Kit must not be used We strongly recommend following the instructions to get robust and reliable results The conformity of the HBV 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 HBV 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 12 SPECIFICATIONS a Analytical specificity the HBV Quantitative Real Time P
9. ed 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 DNA in the sample 3 CONTENT Table 1 PREP NA DNA RNA Extraction Kit Precipitation buffer Colorless liquid Washout solution 1 Colorless liquid 50 ml 1 vial 1 25 mli 5 ml 1 25 ml in Dissolving buffer Colorless liquid each tube 3 ml 1 5 ml in each tube Internal control IC Colorless liquid Table 2 Standards ST1 1x10 copies ml Colorless liquid 75 uL ST2 3x10 copies ml Colorless liquid 75 uL Table 3 HBV Quantitative Real Time PCR Kit Composition of colorless l Mes 1 92 ml 0 02 uL 96 separate 1x96 or Paraffin sealed PCR mix liquid and white waxy Ser iube stripped 812 tubes fractions TECHNO Taq polymerase Colorless viscous liquid 50 uL PCR buffer Colorless liquid 2 tubes Negative control C Colorless liquid 2 tubes 1 ml 0 5 ml in each tube Positive control C Colorless liquid 150 uL 2 ml 1 ml in each tube Mineral oil not supplied 2 in Kit for Rotor Gene tubes Colorless viscous liquid The approximate total time needed to perform the assay is 4 hours The PREP NA DNA RNA Extraction Kit is sufficient for extraction of 100 samples The HBV Quantitative Real Time PCR Kit sufficient to test 44 samples in duplicates 4 REAGENTS AND EQUIPMENT REQUIRED BUT NOT PROVIDE
10. er 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 7 10 7 11 7 12 7 13 Add 5 0 uL of C C 511 and ST2 into corresponding tubes Avoid paraffin layer break Spin tubes briefly 1 3 sec Set the tubes to Real Time PCR Termal Cycler 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 DTprime Thermal Cyclers Optical measurement Type of the PCR Melt Data Acquisition a 39 9 Reime nn nn nn AA 10 Storage Table 6 The PCR program for iCycler iQ5 thermal cyclers with dynamic factor PCR Melt Data St Dwell ti Set PE m Lis iuh Acquisition dynamicwf tmo program 0 A 100301 A A 160 ie Lo 1 J 0320 20950 J ss 2 020 620 RealTime 5 e l 3100 Storage Table 7 The PCR program for Rotor Gene Thermal Cyclers u Take the measurement Table 8 Detection channels Cycle Repeats 1 time 50 times Yellow 8 CONTROLS Table 9 Result The controlled iis step Specific signal is Specific signal is present absent PCR DNA extraction C Interpretation Va
11. lid Invalid Invalid Valid Valid Valid Invalid 10 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 IC is present If the signal for C is present whole tests of current batch considered false Decontamination required 9 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 10096 range The interpretation should be performed in accordance with table 10 Table 10 Detection channel Fam Green Interpretation i Hex Yellow Cp Ct copies ml Test samples Positive with specified viral load 7 5x10 1 0x10 Not considered copies ml 5 Positive with notification Less than Less than 7 5x10 Not considered 750 copies ml no specified value Positive with notification More More than 1 0x10 Not considered than 1 0x10 copies ml no specified value Not specified N A Specified Cp Ct 29 34 Not ified N A C 2 2 10 7 1x10 Not considered Positive with specified viral load copies ml Not specified Specified Cp Ct 29 34 Negative If the concentration of the C falls out the 2 2x10 7 1x10 range th
12. on 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 1 tube for the positive control C For example if you need to test 10 samples mark 22 tubes 20 for the samples 1 for C and 1 for C4 6 4 6 5 Add 10 uL of the premixed IC in each tube Add 300 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 6 6 7 6 8 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 Add 100 uL of the blood plasma sample into the marked tubes Do not add samples to the C and C tubes Add 100 uL of the C and C into corresponding tubes
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