法庭科学SWGDAM验证流程课件.pptx

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1、法庭科学SWGDAM验证流程,议程,SWGDAM 介绍华夏TM白金SWGDAM 验证YfilerPlusTM SWGDAM 验证ISO 18385 介绍,SWGDAM主页,Source: swgdam.org,关于SWGDAM,First meeting of Technical Working Group on DNA Analysis Methods (TWGDAM) was held in November 1988, with a subcommittee on restriction fragment length polymorphism (RFLP) DNA analysis.

2、TWGDAM continued to provide a level of direction to the forensic DNA community by issuing guidelines for DNA analysis, and the guidelines became de facto standards and were recognized by courts.SWGDAM Chairman is appointed by the FBI Director, and members are selected by the Chairman from forensic D

3、NA laboratories.SWGDAM has multiple committees including Autosomal STR Committee, CODIS Committee, Rapid DNA Committee, Next Generation Sequencing Working Group, etc.One of SWGDAMs most important responsibilities is the recommendation of revisions to the FBIs Quality Assurance Standards (QAS) for DN

4、A Analysis.,SWGDAM 出版物,SWGDAM法医DNA分析方法验证指导,3.7 Population Studies,3.8 Mixture Studies,3.9 PCR Based Studies,3.3 Sensitivity,3.4 Stability,3.5. Precision & Accuracy,3.6 Case Type Samples,3.2 Species Specificity,3.1 Genetic Markers,SWGDAM Validation Guidelines for Forensic DNA Analysis Methods approve

5、d in November 2012 www.swgdam.org,华夏白金物种特异性 非灵长类,10 ng non-primate DNA, or 105 copies of microbes (species commonly found in the oral cavity, listed in User Guide) were added to the system using 29 PCR cycles.Low peaks were detected with bovine, dog (like contamination, also found in the repeat), ho

6、rse, mouse and pig.,Bovine,Chicken,Dog,Horse,Microbes,Mouse,Pig,Rabbit,Rat,Sheep,NTC,200 rfu,200 rfu,700 rfu,SWGDAM 3.2 The ability to detect genetic information from non-targeted species (e.g., detection of microbial DNA in a human assay) should be determined.,华夏白金一次性成功通过率,All sample types have 90%

7、 pass rate (PAT = 175 rfu) with 1 Proportion test.The recommended rxn volume for HXP is 25ul, some tests here were done with 10ul reaction volume to challenge the system.,SWGDAM 3.4 The ability to obtain results from DNA recovered from biological samples deposited on various substrates and subjected

8、 to various environmental and chemical insults should be evaluated.,样本准在3500 xl上的准确度,Size deviation of 42 population samples showed deviation 0.50bp for all alleles,SWGDAM 3.5 Precision and accuracy of the assay should be demonstrated. Accuracy is the degree of conformity of a measured quantity to i

9、ts actual (true) value.,遗传标记在不同种族的人群中的分布,SWGDAM 3.7 The distribution of genetic markers in populations should be determined in relevant population groups.,Allele frequency for major Chinese population groups was studied.Data of D6S1043 was presented above, and the data with other markers was publish

10、ed in UG.,反应预混物保护带研究,Eight raw materials in master mix were varied up to 20%. ICB was above 35% for most raw materials with 10% or even 20% variation.Detail data including capability analysis presented next.,Component concentrations,Individual components,The Huaxia Platinum MM is robust to raw mater

11、ial change,SWGDAM 3.9 The reaction conditions needed to provide the required degree of specificity and robustness should be determined.,SWGDAM 对Y-STR的指导,Y-STR typing is also used in lieu of autosomal typing for the detection of male DNA in mixtures that contain an overabundance of female DNA. Given

12、that under certain conditions a male minor contributor in a mixture of female:male DNA may only be detectable by Y-STR typing, laboratories should pursue Y-STR analysis as the most appropriate means of detecting a male contributor(s) in some forensic samples,Interpretation Guidelines for Y-Chromosom

13、e STR Typing approved in January 2014 www.swgdam.org,This SWGDAM statement highlights the importance of male specificity of Y-STR kit with the male female mixtures commonly found with the sex assault cases.,Primer mixDesign/screen primer pairs with minimal female cross-reactivityMaster mixLower the

14、Mg and Taq enzyme concentrations to minimize female cross-reactivityMultiple rounds of DOE were performed to optimize the MM componentsPCR conditionIncrease the annealing/extension temperature to increase male specificity,提高Y-特异性的工作,Yfiler Plus 在男女混合DNA的表现,Yfiler Plus recovered full profile with 1:1

15、000 male:female mixtureNo extra peaks with Yfiler Plus, while multiple extra peaks with PPY23 and Yfiler,Internal Product Spec: The kit will recover full profile with average 40% ICB when amplified with male:female mixtures of 1:1000,Yfiler Plus高浓度女性DNA下的基线,200rfu,Low female cross-reactivity (175 PA

16、T) within the read region are all documented in the UG.,Blue 271,Red 139/144,Red grass,Outside the read region,68 bp,406 bp,Internal Product Spec: The Yfiler Plus kit shall not amplify reproducible female DNA in the 1 ug range, without reproducible drop ins & artifacts within the read region.,3 微克女性

17、DNA在三个盒子的比较,3 ug of DNA from 10 different female donors were tested with the Yfiler Plus, PPY23 and Yfiler kits. The highest reproducible female artifacts from each sample were plotted by kit. Yfiler Plus and Yfiler have significantly lower artifact peak heights than PPY23.,PAT,新的法医DNA标准 ISO 18385,Meet ISO 18385 requirements for the manufacture of Forensic DNA Grade reagents,Provide same level of manufacturing quality and QC testing across HID product lines,Both Yfiler plus and Huaxia Platinum kits are ISO 18385 compliant,Manufacturing Quality Commitment,