PG1502 DNA Detection Kit User`s Manual Pharmigene Inc. 1502C
Contents
1. PG1502 DNA Detection Kit CE 0120 REF PG 1502C 024 FE PG 1502C 048 PG 1502C 096 For In Vitro Diagnostic use 1 Intended Use The intended use for PG1502 DNA detection kit is to detect the presence of HLA B 1502 allele in the blood samples of genetically at risk populations 2 Materials 2 1 Kit contents Reagent Content and functions 96 48 24 Rxn Rxn Rxn 10x conc reagent containing the mix of forward and reverse primers for HLA B 1502 allele Use to amplify the specific region of human HLA B 1502 allele 10x conc reagent containing the mix of forward and reverse primers for internal control gene 120 ul 60 ul Use to amplify the specific region of internal control gene Include the fragment of HLA B 1502 allele and 64 ul 32 ul Internal control gene 1 2ml 1 2ml 1 5 ml tube with cap 96 well PCR optical plate Optical adhesive film Genotype Detection 2x conc reagent containing SYBR GREEN I dye Hot start DNA polymerase dNTPs ROX reference dye and buffer Use to adjust the final PCR O Nuclease Free reaction volume Clear Water used as No template Control NTC 2 2 Material required but not provided 10 ul micro pipette 20 ul micro pipette 200 ul micro pipette 2 3 Instrument required but not provided Real time PCR detection system selected from ABI 7500 7000 Bio rad 1Q5 Cepheid SmartCycler Corbett Rotor Gene 3002. The preparation process should be kept under 4 C Solution A amp B Based on the number of reactions calculated the required amount of the three components shown in the table Then pipette the following components into a 1 5ml Vial Component Solution A Solution B conc 2x PCR Master Mix 12 541 12 541 10x Genotype Detection Mix Zu i 10x Internal Control 2 5ul detection Mix a Nuclease free water Sul Sul gt Total volume 23ul 23ul Mix by vortex and spin down briefly Add 23 ul solution A into the red area and solution B into green area See 7 1 Sample loading chart Add 2 wl of sample DNA into two wells one in red area and the other in green area Apply similar procedures to PC and NTC For PC add 2 ul positive control template blue cap vial For NTC add 2ul nuclease free water clear cap vial 3 Seal the PCR plate with an optical adhesive film Quick centrifuge at 1500 rpm for 10 sec Place the PCR plate in real time PCR instrument Instrument setup is described in 4 3 2 1502C 20100426 Page 1 of 2 4 4 Data Analysis Model of Real Time PCR system ABI 7500 Fast Real time PCR System ABI 7000 Sequence Detection System After the PCR reaction the value of threshold should be set manually for the PCR machine you use The threshold value on the instrumental system is listed below Threshold 0 15 0 15 Bio Rad iCycler 1Q5 Real Time PCR Detection System Cepheid SmartCycler System Corb
3. Dissociation Curve 3 200 E1 2 700 E1 2 200 E1 1 700 E1 Derivative 1 200 E1 7 000 E2 2 000 E2 3 000 E2 60 0 65 0 70 0 75 0 80 0 85 0 90 0 95 0 Temperature C ead PharmiGene Inc 9F 3 No 21 Lane 169 Kangning St Xizhi City Taipei County 22180 886 2695 9800 Obelis S A Av De Tervueren 34 bte 44 1040 Brussels Belgium Notice to Purchaser a 2010 Pharmigene Inc All rights reserved b This product is licensed under U S Patent No 7 470 513 or corresponding foreign patents which assigned to Pharmigene Inc Other patents are pending c PHARM I GENE and PHARMIGENE are registered trademarks of Pharmigene Inc Warranty and Liability Pharmigene is committed to providing the highest quality reagent at competitive prices Pharmigene warrants that the products meet or exceed the performance standards described in the product specification sheets If you are not completely satisfied with any product our policy is to replace the product or credit the full purchase price and delivery charge No other warranties of any kind expressed or implied are provided by Pharmigene Pharmigene s liability shall not exceed the purchase price of the product Pharmigene shall have no liability for direct indirect consequential or incidental damages arising from the use results of use or inability to use its Products 1502C 20100426 Page 2 of 2
4. 0 or Roche LC480 DNA extraction system Qiagen QIAamp DNA Mini Kits is recommended 3 Introduction 3 1 Background HLA B 1502 allele is found to have a strong linkage with Carbamazepine induced Steven Johnson Syndrome SJS and Toxic Epidermal necrolysis TEN in Asian descendents HLA B 1502 allele is located in human chromosome 6 and belongs to human leukocyte antigen The results of PG 1502 DNA detection can be used as an aid for the clinicians to identify the patients with the presence of HLA B 1502 allele that may pose a greater risk of carbamazepine CBZ induced SJS TEN 3 2 Component Description PG1502 DNA detection kit contains HLA B 1502 allele specific primers controls internal control mix PCR master mix and nuclease free water 3 3 Number of Test Package with size of 96 reactions can be used to conduct a maximum of 46 specimens size with 48 reactions for a maximum of 22 specimens and size with 24 reactions for maximum 10 specimens The final volume of each reaction is 25ul 3 4 Storage Stability The kits should be stored at 15 20 C and the unopened kit can be kept stable until the expired date The expired date is 6 months after manufactured date Stored at 4 C is not recommended Do not subject the kit more than 4 freeze thaw cycles If the kits are not often to be used aliquot and freeze the reagent is recommended 3 5 Analytical performance 516 blood samples were tested to obtain the performance of PG1502
5. DNA detection kit The positive percent agreement of PG1502 DNA detection kit is greater than 99 and the negative percent agreement is 92 39 The Pharmigene Inc Package size Volume PG1502 DNA Detection Kit User s Manual overall percent agreement between PG1I502 DNA detection kit and sequence based typing is 93 41 3 6 Sample collection and handling Technician should wear safety equipments while handling the samples The whole peripheral blood should be collected in a tube containing either sodium citrate or K2 EDTA as the anticoagulants Do Not Use the Heparin as the anticoagulant Do Not Use hemolyzed blood sample It is highly recommended that DNA extraction should be performed either at the day of blood sample collection when stored in 4 C The assay should be performed as early as possible after DNA extraction and no later than 7days of storage at 4 C 3 7 Interference According to laboratory data the performance was not affected for the blood sample containing bilirubin less than 8mg dL lipid less than 150mg dL or Aspirin less than 35ug mL However decrease performance was observed with DNA samples containing 0 025 of hemoglobin and very high residual Qiagen wash buffer 4 Protocol 4 1 HLA B 1502 detection and Internal control detection IC Two independent PCR reactions should be conducted simultaneously for each DNA sample One reaction is for HLA B 1502 allele detection add Genotype Detection Mix of red cap
6. ett Research Rotor Gene 3000 Two Ct values for each sample will be obtained one from Genotype Detection Mix and the other from Internal Control Detection Mix The differences of Ct values are calculated according to the equation shown below ACt Ct Genotype Detection mix Ct Internal control detection mix When the Ct value of internal control is equal to or less than 27 and the ACt value is equal to or less than 7 the result should be identified as HLA B 1502 allele positive Whereas the ACt value is greater than 7 the result should be identified as HLA B 1502 allele negative When the Ct value of internal control is greater than 27 the PCR inhibition should be suspected and repeating the test is highly recommended 4 4 1 Result identification chart IC Ct lt 27 PCR inhibitors may be present in specimen IC Ct gt 27 HLA B 1502 positive HLA B 1502 negative HLA B 1502 negative Ct Genotype Detection Mix lt 35 Ct Genotype Detection Mix gt 35 undetermined Retest Inappropriate Inappropriate gDNA quantity quantity IC internal control 4 4 2 The results of PC and NTC should be confirmed to be acting normal before the sample data is processed For PC the Ct value of internal control should be equal to or less than 27 and ACt value should be equal to or less than 7 For no template control the result should be undetermined or Ct gt 35 4 4 3 If there is any question about data interpre
7. tations please call PharmiGene service line 886 02 2695 9800 5 Warning and Caution A AD Ta Technician should wear safety equipments Warning All products contain blood derivations should be treated as potentially infectious No known test or method can confirm that human blood derivations will not transmit infectious material These product components may cause irritant Avoid contacting with eyes Skin or cloth If contacting with eyes skin or mucous wash immediately with water aAfter experiment completely clean the operation area and flush the splash area with plenty water If the test is interrupted repeat the test to collect the correct result The Result can only be used as genotype identification The use of Carbamazepine should be judged by clinical professionals References 1 Nature Vol 428 p 486 2004 2 Pharmacogenetics and Genomics Vol 16 p 297 306 2006 Appendix Sample loading chart Pharmigene Inc etection Kit User s Manual 9 10 11 12 PG1502 DNA D 7 crQaayarmAonk wp PC positive control NTC no template control Red area for HLA B 1502 allele detection Green area for internal control gene detection Amplification Plot 1 000 Exp 1 1 000 j 1 000 B14 i 4 1 000 E2 1 000 3 i 1 000 B44 Detector SYBR Plot ARn vs Cycle Threshold 0 15511833 Dissociation Plot
8. vial and the other is for internal control gene detection add Internal Control Detection Mix of green cap vial The purpose of internal control gene detection is to identify the false negative result caused by PCR inhibition 4 1 1 No template control NTC No template control should be tested in each run When testing use the supplied nuclease free water clear cap vial to replace the sample DNA 4 1 2 Positive control PC Positive control should be tested in each run When testing use the supplied positive control template to replace sample DNA 4 2 Sample DNA Add 2ul of sample DNA per reaction DNA amount should be within 25 100 ng reaction The sample DNA will be extracted from human whole blood of the patient using the recommended DNA extraction system See 2 3 The sample DNA OD260 280 ratio should be adjusted to between 1 7 and 2 0 using spectrophotometer 4 3 Procedure 4 3 1 Defrost the reagent on ice Gently mix Place the reagent on ice after a quick centrifuge Do Not Mix Different Lots in the Same Run 4 3 2 Set real time PCR system amplification program and add dissociation curve stage as below Time Temp Cycle Description 1 DNA polymerase l BOSE Te a cis 10 min 95C 1 polymerase is activated by this step 2 Amplification cycle 35 Denaturation 15 sec 95 C Fluorescence signal is Annealing Extension 1 min Tae collected in this step in each cycle add dissociation curve stage 4 3 3 Master Mix Preparation
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