Uterine Cervix Cancer of High-risk HPV Genotype Related
Contents
1. cler I DNA sample template positive and negative controls to different reaction plate tubes Immediately close the plate tubes to avoid contamination 3 Spin down briefly in order to collect the Master Mix in the bottom of the reaction tubes 4 Perform the following protocol in the instrument 94 C for 2min Target Nucleic Acid 93 C for 10sec 62 C for 31sec igeceiee Fluorescence measured at 60 C y HEX VIC JOE Target Nucleic Acid 5 A If you use ABI Prism system please choose none as passive reference and quencher 10 Threshold setting just above the maximum level of molecular grade water 11 Quality control Negative control and positive control must be performed correctly otherwise the sample results is invalid Molecular Grade Water Positive Control Negative control Channel HPV 16 56 Reaction Mix UNDET UNDET Ct lt 35 HPV 18 45 Reaction Mix UNDET UNDET Ct lt 35 HPV 35 59 Reaction Mix UNDET UNDET Ct lt 35 UNDET C35 C35 HPV 58 52 Reaction Mix HPV 31 IC Reaction Mix UNDET UNDET ct lt 35 HPV 33 Reaction Mix UNDET UNDET ct lt 35 HPV 68 Reaction Mix UNDET UNDET ct lt 35 12 Data Analysis and Interpretation 1 The Ct value shows lt 38 The result is positive The sample contains some serotype of HPV DNA The following results are possible HPV 16 56 HPV 31 IC xvie SS mva ss m S OSOS Hve _ mexmicnoe O O O O 2 T2. he Ct value shows 38 40 please repeat again If the result still shows 38 40 it can be considered negative But the clinical samples in channel of HEX VIC JOE in HPV 31 IC Reaction Mix should be positive Otherwise the negative result of the sample is invalid Please refer to section 9 2 in detail 3 In channel FAM or HEX VIC JOE no signal is detected in any one of 13 Serotypes HPV Master Mix The sample does not contain any one of 13 Serotypes HPV It can be considered negative For further questions or problems please contact our technical support at trade liferiver com cn Selection of fluorescence channels HPV 18 45 HPV 35 59 HPV 39 51 HPV 58 52
3. l contained all 13 types of high risk HPV partial sequence 4 Kit Contents Type of Reagent DNA Extraction Buffer 1 vial 1 8ml HPV 16 56 Reaction Mix 1 vial 950ul HPV 18 45 Reaction Mix 1 vial 950ul HPV 35 59 Reaction Mix 1 vial 950ul HPV 39 51 Reaction Mix 1 vial 950ul HPV 58 52 Reaction Mix 1 vial 950ul HPV 31 IC Reaction Mix 1 vial 950ul HPV 33 Reaction Mix 1 vial 950ul HPV 68 Reaction Mix 1 vial 950ul PCR Enzyme Mix 1 vial 88ul Molecular Grade Water 1 vial 400ul HPV Positive Control 1 vial 100ul Analysis sensitivity 5X 10 copies ml Note Analysis sensitivity depends on the sample volume elution volume nucleic acid extraction methods and other factors If you use the DNA extraction buffer in the kit the analysis sensitivity is the same as it declares However when the sample volume is dozens or even hundreds of times greater than elution volume by some concentrating method it can be much higher 5 Storage e All reagents should be stored at 20 C Storage at 4 C is not recommended e All reagents can be used until the expiration date indicated on the kit label e Repeated thawing and freezing gt 3x should be avoided as this may reduce the sensitivity of the assay e Cool all reagents during the working steps e Super Mix should be stored in the dark 6 Additionally Required Materials and Devices e Biological cabinet e Vortex mixer e Cryo container e Sterile filter tips for micro pipets e Disposable gloves
4. powderless e Biohazard waste container e Refrigerator and Freezer e Tube racks e Desktop microcentrifuge for eppendorf type tubes RCF max 16 000 x g 7 ASwarnings and Precaution e Carefully read this instruction before starting the procedure e For in vitro diagnostic use only e This assay needs to be carried out by skilled personnel e Clinical samples should be regarded as potentially infectious materials and should be prepared in a laminar flow hood e This assay needs to be run according to Good Laboratory Practice e Do not use the kit after its expiration date e Avoid repeated thawing and freezing of the reagents this may reduce the sensitivity of the test e Once the reagents have been thawed vortex and centrifuge briefly the tubes before use e Prepare quickly the Reaction mix on ice or in the cooling block e Set up two separate working areas 1 Isolation of the RNA DNA and 2 Amplification detection of amplification products e Pipets vials and other working materials should not circulate among working units e Use always sterile pipette tips with filters e Wear separate coats and gloves in each area e Do not pipette by mouth Do not eat drink smoke in laboratory e Avoid aerosols 8 Sample Collection Storage and Transport e Collect samples in sterile tubes e Specimens can be extracted immediately or frozen at 20 C to 80 C e Transportation of clinical specimens must comply with local regulations for the transpor
5. rcial kit depending on the yield For DNA extraction please comply with the manufacturer s instructions 9 2 Internal control MNBH gene is detected as an internal control All clinical samples should exhibit MNBH positive thus indicating the presence of sufficient nucleic acid from human MNBH gene Failure to detect MNBH in any of clinical samples may indicate that 1 Improper extraction of nucleic acid 2 Absence of sufficient human cellular material in sample 3 Improper assay set up and execution 4 Reagent or equipment malfunction 9 3 PCR Protocol The Master Mix volume for each reaction should be pipetted as follows 3Gp Dyl 22 5 0 4ul Reaction Mix Enzyme Mix Reaction Mix Enzyme Mix Apl 22 9ul Master Mix Master Mix 4ul 36ul 2 5 22 5 Extraction DNA Master Mix Extraction DNA Master Mix Reaction Reaction Plate Tube Plate Tube fl fl This system is onhy for PCR Instrument OR PCR Instrument cant Cycler Il 1 The volumes of Reaction Mix and Enzyme Mix per reaction multiply with the number of samples which includes the number of controls standards and sample prepared Molecular Grade Water is used as the negative control For reasons of unprecise pipetting always add an extra virtual sample Mix completely then spin down briefly in a centrifuge 2 Pipet 36u 22 51 for SmartCycler II Master Mix with micropipets of sterile filter tips to each real time PCR reaction plate tubes Separately add 4pl 2 5ul for SmartCy
6. rescence signal is detected initially is proportional to the amount of the specific PCR product Monitoring the fluorescence intensities in real time allows the detection of the accumulating product without having to re open the reaction tube after the amplification 3 Product Description The human papilloma virus HPV is one of the most common virus groups in the world to affect the skin and mucosal areas of the body Different types of the HPV s are known to infect different parts of the body 13 types of genital tract HPV in particular HPV 16 18 31 33 35 39 45 51 52 56 58 59 68 are known to cause up to 99 of cervical cancers and new studies show that they may be linked to oral cancer as well All of these are genital viruses which are spread through sexual contact Uterine Cervix Cancer of High risk HPV Genotype Related Real time PCR kit contains a specific ready to use system for the detection of 13 Types of High risk HPV Genotypes in genital swabs samples by polymerase chain reaction in the real time PCR system The master contains reagents and enzymes for the specific amplification of HPV DNA Fluorescence is emitted and measured by the real time systems optical unit during PCR The detection of amplified HPV DNA fragment is performed in fluorimeter channel FAM and HEX VIC JOE with the fluorescent quencher BHQ1 DNA extraction buffer is available in the kit and genital swabs samples are used for DNA extraction An external positive contro
7. t of 1 2 3 4 5 6 7 8 9 e Real time PCR system e Real time PCR reaction tubes plates e Pipets 0 5ul 1000n1 e Sterile microtubes etiologic agents 9 Procedure 9 1 DNA Extraction DNA extraction buffer is supplied in the kit please thaw the buffer thoroughly and spin down briefly in the centrifuge before use You may use your own extraction systems or commercial kits 1 Wash the genital swabs in 1 0ml normal saline and vortex vigorously Centrifuge at 13000rpm for 5 minutes Carefully remove and discard supernatant from the tube without disturbing the pellet 2 Add 1 0ml normal saline and suspend the pellet with vortex vigorously Centrifuge at 13000rpm for 5 minutes Carefully remove and discard supernatant from the tube without disturbing the pellet 3 Add 50ul DNA extraction buffer close the tube then vortex for 10 seconds Spin down briefly in a table centrifuge 4 Incubate the tube for 10 minutes at 100 C 5 Centrifuge the tube at 13000rpm for 5 minutes The supernatant contains the DNA extracted and can be used for PCR template Attention A During the incubation make sure the tube is not open Since the vapor will volatilize into the air and may cause contamination if the sample is positive B The extraction sample should be used in 3 hours or stored at 20 C for one month C DNA extraction kits are available from various manufacturers You can also use your own extraction systems or the comme
8. wm ZJ Bio Tech Co Ltd www liferiver com cn Tel 86 2 1 34680596 trade liferiver com cn Fax 86 21 34680595 2 floor No 15 Building No 188 Xinjunhuan Road PuJiang Hi tech Park Shanghai China Liferiver Issue Date Sep 6 2012 C Uterine Cervix Cancer of High risk HPV Genotype Related User Manual For In Vitro Diagnostic Use Only For use with ABI Prism 7000 7300 7500 7900 Step One Plus iCycler iQ 4 iQ 5 Smart Cycler Il Bio Rad CFX 96 Rotor Gene 6000 Mx3000P 3005P MJ Option2 Chromo4 ec Ea Obelis S A Boulevard G n ral Wahis 53 Tel 32 2 732 59 54 Fax 32 2 732 60 03 Revision No ZJ0010 Real Time PCR Kit 20 C TD 0031 02 r LightCycler 480 Instrument 1030 Brussels BELGIUM E Mail mail obelis net 1 Intended Use Uterine Cervix Cancer of High risk HPV Genotype Related Real time PCR kit is used for the detection of 13 Types of High risk HPV Genotypes in genital swabs samples by using real time PCR systems 2 Principle of Real Time PCR The principle of the real time detection is based on the fluorogenic 5 nuclease assay During the PCR reaction the DNA polymerase cleaves the probe at the 5 end and separates the reporter dye from the quencher dye only when the probe hybridizes to the target DNA This cleavage results in the fluorescent signal generated by the cleaved reporter dye which is monitored real time by the PCR detection system The PCR cycle at which an increase in the fluo
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