HBV PCR detection Kit USER MANUAL
Contents
1. 7 5 7 6 Tad 7 8 7 9 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 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 DNA sample 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 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 Add 5 uL of C which passed whole DNA extraction procedure 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 1 6 to refer the cycling program and table 7 to2. and DNA o t i O a CU y PCR and DNA C extraction faid o i IC DT 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 11 9 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 8 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 The interpretation should be performed in accordance with table 9 10 Table 9 Results with HBV Conventional PCR detection Kit Specific product 295 bp Internal control 560 bp Not considered Positive Positive Negative Table 10 Results with HBV Flash and Real time PCR detection Kit HBV FLASH PCR Test samples d
3. 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 nucleic acid preparation is ready The preparation can be stored at 20 C for 1 month or at 70 C for 1 year 7 PCR PROTOCOL 7 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 samples 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 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 N Hold TECHNO Taq polymerase at room temperature as short time as possible The overheating is detrimental to its performance 7 4 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 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
4. refer the detection channels applicable to Real Time PCR kits Using Thercyc cycler you need to choose Precision active regulation regulation algorithm Table 1 The PCR program for Tercyc Conventional PCR Thermal Cycler applicable to Conventional PCR kits Step For thermal cyclers with active regulation Number of cycles T 94 64 72 94 64 72 Storage Table 2 The PCR program for Tercyc Conventional PCR Thermal Cycler applicable to FLASH PCR kits Step For thermal cyclers with active regulation Number of cycles Temperature Time x 9 5 0 94 _ 64 67 94 64 67 5 Table 3 The PCR program for DT ite and DT prime Thermal Cyclers Optical measurement Type of the 00 20 E e Table 5 The PCR program for iCycler iQ5 thermal cyclers with dynamic factor PCR Melt Data R St Dwell ti Set tee MUR as diii s Acquisition dynamicwf tmo program ss T 13 999 399 WeWme EN 5 hA h J j Lo 1 J 0320 HP x 2 020 620 RealTime 5 l 3100 J Storage 10 Table 6 The PCR program for Rotor Gene Thermal Cyclers Cycling Hold Time Cycle Repeats 94 C Cycling 2 38 C 50 times 62 C Take the measurement Table 7 Detection channels 8 CONTROLS Table 8 Result The controlled UNE l eee l Control t Specific signal is Specific signal is Interpretation P present absent x Invalid PCR
5. Detection Kit endpoint HBV FLASH PCR Detection Kit and HBV Conventional PCR Detection Kit The HBV 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 HBV FLASH PCR Detection Kit is based on the same principle but the fluorescence is measured only once after reaction HBV 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 DTlite or DTprime REAL TIME Thermal Cyclers for HBV 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 REF ec cu O GENE4 EU for HBV FLASH PCR Detection Kit The HBV Real Time PCR Detection Kit is also approved for use with iQ Bio Rad Laboratories and Rotor Gene Qiagen real time thermal cyclers The HBV FLASH PCR Detection Kit is also approved for use with Ala1 4 fluorescence reader BioSan DNA extraction On this step the internal control sample
6. 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 PCR detection 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 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 For example if you need to test 10 samples mark 11 tubes 10 for the samples 1 for C 6 4 6 5 Add 10 uL of the premixed internal control DNA 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 b
7. For professional use only HBV 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 P602 23 9EU F1 P602 52 1bEU R1 P602 S3 9EU F1 P602 21 1EU 184 1 20 03 13 REF R1 P602 24 9EU E3 P602 50 1EU VER F1 P602 51 1EU 3 P602 20 1EU 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 PCR detection Kit is intended for research and diagnostic applications The HBV PCR detection Kit is an in vitro Nucleic Acid Test NAT based pathogen detection product The HBV PCR detection Kit is designed to detect Hepatitis B Virus HBV nucleic acids in human blood plasma The HBV PCR detection Kit can be used in clinical practice for HBV diagnostics 2 METHOD The implemented PCR method is based on amplification of a target DNA sequence The detection can be performed in each of three variants real time HBV Real Time PCR
8. IC is added to the samples It is needed for test quality assurance 3 CONTENT Table 1 PREP NA DNA RNA Extraction Kit K 2 buffer Colorless liquid solution 1 Colorless liquid 50 ml 1 vial 1 25 ml i m i 4 tubes Dissolving buffer Colorless liquid each tube 3 ml 1 5 ml in each tube Internal control DNA IC Colorless liquid Table 2 HBV PCR detection Kit Color coded composition of liquid 1 92 ml or 2 0 ml and white waxy 0 02 mL per tube fractions TECHNO Taq polymerase Colorless viscous liquid 50 uL PCR buffer Colorless liquid 2 tubes Negative control C Colorless liquid 2 tubes 96 or 100 separate or stripped tubes of 0 2 or 0 5 ml Paraffin sealed PCR mix 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 MN Colorless viscous liquid in Kit for Rotor Gene 4 The approximate total time needed to perform the assay is 4 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 HBV PCR detection Kit sufficient to test 96 100 samples including negative positive and internal controls 4 REAGENTS AND EQUIPMENT REQUIRED BUT NOT PROVIDED 4 1 Specimen collection The whole blood samples shoud
9. 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 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 HBV Real Time PCR Detection Kit Tercyc Conventional PCR Thermal Cycler REF o cu or equivalent for HBV FLASH PCR Detection Kit and HBV Conventional PCR Detection Kit Gene or Gene 4 Fluorescence Reader a O GENE4 EU or Ala1 4 fluorescence reader or equivalent for HBV FLASH PCR Detection Kit For electrophoretic detection for HBV Conventional PCR Detection Kit 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 microorganisms of I IV classes of pathogenicity Handle and dispose all biological samples
10. efore 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 6 22 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 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 Ne2 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 uL of the dissolving buffer to the precipitate Spin down the
11. etection Kit HBV Real time PCR detection Kit Interpretation Hex Yellow Cp Ct is specified Not considered Cp not specified for iQ N A igi Mi uncertain Cp not specified Cp not specified for O 1 Stern A SMA incar x CG Positive _ a Cp not specified i for iQ N A Cp Ct 29 34 Negative if Cp Ct HEX more than specified the result is invalid 12 10 TROUBLESHOOTING Table 10 Operation error p Repeat whole test PCR inhibition Violation of storage anq i Dispose current batch handling requirements Dispose current batch Perform decontamination procedures If you face to any undescribed issues contact our representative Contamination 11 STORAGE AND HANDLING REQUIREMENTS Expiry date 9 month from the date of production All components of the HBV 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 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 An expired HBV PCR detection Kit must not be used We strongly recommend following the instructions to get robust and reliable results The conformity of the HBV PCR detection Kit to the prescribed technical requirements is subject to compliance of storage carriage a
12. nd handling conditions recommended by manufacturer Contact our customer service by quality issues of the HBV 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 12 SPECIFICATIONS a Analytical specificity the HBV PCR detection Kit allows detection of all known HBV subtypes The samples containing HBV will be defined as positive The samples not containing HBV will be defined as negative b Sensitivity not less than 200 copies of HBV DNA per 1 ml of blood plasma 13 c Diagnostic sensivity 99 896 d Diagnostic specificity 10096 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 14 14 KEY TO SYMBOLS O lt ITI AJ s EYE Caution Consult instructions for use Date of manufacture Expiration date In vitro diagnostic medical device Batch code Version ONTROL u V n Manufacturer Negative control Positive control Cataloque number Sufficient for Temperature limitation Upper limit of temperature 15 16
13. 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 kit and those recommended by manufacturer Do not mix reagents from different batches
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