《应用dhplc技术进行诊断性分析的质量保证体系》由会员分享，可在线阅读，更多相关《应用dhplc技术进行诊断性分析的质量保证体系（65页珍藏版）》请在金锄头文库上搜索。
1、应用DHPLC技术进行诊断性分析的质量保证体系,张泽云 美国环球基因公司中国代表处,诊断性分析的要求,临床分子遗传学分析的复杂性 临床分子检测结果的一致性和精确性 变性高效液相色谱(DHPLC)作为一种高效和敏感的基因突变检测技术 DHPLC技术质量控制,AMERICAN COLLEGE OF MEDICAL GENETICS Standards and Guidelines for Clinical Genetics Laboratories 2005 Edition G: CLINICAL MOLECULAR GENETICS These Standards and Guidelines
2、specifically refer to the use of molecular techniques to examine heritable or somatic changes in the human genome. G18 Denaturing High Performance Liquid Chromatography (dHPLC) (Section Added November 2003),CMGS Best Practice Guidelines Use of the WAVE System in Diagnostic Service Prepared and edite
3、d by John Harvey, National Genetics Reference Laboratory (Wessex), Salisbury, UK and Els Schollen, Centre for Human Genetics, Leuven, Belgium last update: 12 March 2004 Introduction Laboratory process DHPLC system Data quality Checking & reporting guidelines References,DHPLC SOPs,Instrument or maint
4、enance SOP Technique General DHPLC SOP WAVE 3500, 3500 HT Method Disease-specific SOPs Rett, BRCA, HNPCC Marfan, Application,company + users,general users + company,specific users,Supplementary Appendix 1 STANDARD OPERATING PROCEDURE WAVE System Operation and Maintenance SOP- O&M WAVE System Operati
5、on and Maintenance For WAVE System Models 3500, 3500A and 3500HT,WAVE System Operation and Maintenance,Analysis of the WAVE Low & High Range Mutation Standards The maintenance procedure DNASep and DNASep HT cartridge maintenance,Role of Mutation standards: checking of correct functioning of the WAVE
6、 System, including oven calibration, cartridge performance, buffer composition and stability, to ensure reproducibility and accuracy of the chromatographic analysis. Mutation standards be run when: l The routine pre-run, l Weekly and monthly maintenance procedure, l After replacement of any componen
7、t, l Validation for a new batch, l As an assay control, at the beginning and end of every run, preferably also after every 100 injections for long runs.,Analysis of the WAVE Low & High Range Mutation Standards,Normal ranges of the mutation standards,The maintenance procedure,3.1 Filter and flush 3.2
8、 Pre-run maintenance 3.3 Weekly maintenance 3.4 Quarterly maintenance 3.5 Other maintenance operations 3.6 Preventative maintenance procedure and system validation,Filter and flush,The principle of filtration involves preventing unwanted contaminants from entering the system. Filtration applies to t
9、wo specific areas: solvent filtration and in-line filtration. The system flushing is to remove mobile phase salt components that can precipitate under strong solvent conditions.,Pre-run maintenance,1. Buffer check 2. Injection system washing 3. Pressure check 4. Check the absorbance on the detector
10、5. Purge the lines,Weekly maintenance,Inline filter replacement Check the syringe,Quarterly maintenance,Check UV lamp UV lamp replacement Cleaning the system (Isopropanol cleaning),DNASep and DNASep HT cartridge maintenance,1. Regular maintenance schedule Every 96-192 injections: Extended hot wash E
11、very 1000 injections: Reverse hot wash DNASep wash (if reverse hot wash fails to resolve mutation standards) 2. Short-term cartridge storage 3. Long-Term cartridge storage 4. New cartridge installation,Daily Maintenance,Equilibrate the cartridge 50%A 50%B for 15 minutes Run 1-2 blanks Verify system
12、performance (pre-analysis) Run a standard Run Samples Verify system performance (post analysis),Weekly Maintenance,An Extended Active Clean Wash is recommended every 100 injections. (Usually done after each 96 well plate),15-30 minutes,WASH,EQUILIBRATE,45-90 minutes,Run Standards to Verify System Pe
13、rformance!,1000 Injection Maintenance,A Reverse Hot Wash is recommended every 1000 injections,Turn off the pump Reverse the cartridge direction. Set the oven to 80 C. Set the pump to 100% D.,60-90 minutes,Storing the Cartridge,Flush the cartridge with 100%D Buffer. Remove the cartridge from the WAVE
14、 System. Cap the cartridge with end plugs. Store the cartridge at room temperature.,Installing a New Cartridge,Stop the pump flow and remove the old cartridge. Remove the plugs from the new cartridge. Install the new cartridge with the arrow pointing toward the rear of the oven.,Make sure the oven h
15、eats up to at least 40 C. Set the pump to 100%D 0.500mL/min. Ensure the pressure is stable and gradually increase the flow (0.9mL/min or 1.5mL/min). Flush the cartridge for 15 minutes. Set the pump to 50% Buffer A, 50% Buffer B and equilibrate for 30 minutes.,Equilibrating a New Cartridge,Verify New
16、 Cartridge Performance,Low-Range Mutation Standard,DNA Sizing Control Standard,Supplementary Appendix 2 STANDARD OPERATING PROCEDURE DHPLC SOP-DHPLC DHPLC mutation detection on Transgenomic WAVE System 3500 Wavemaker 4.1.44 & HSM 3.0-2.1 (build 2) Navigator 1.5.4 (build 19),PCR requirements,Primer design Template purity and concentration DNA Polymerases PCR buffer mix PCR plates PCR quality and Product mixing Post-PCR, film use Co
