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法庭科学SWGDAM验证流程
钟昌
Staff Scientist | ThermoFisher Scientific
Nov 17th, 2016
The world leader in serving science
议程
• SWGDAM 介绍
• 华夏TM白金SWGDAM 验证
• YfilerPlusTM SWGDAM 验证
• ISO 18385 介绍
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SWGDAM主页
Source: swgdam.org
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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.
• 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 DNA laboratories.
• SWGDAM has multiple committees including Autosomal STR
Committee, CODIS Committee, Rapid DNA Committee, Next Generation
Sequencing Working Group, etc.
• One of SWGDAM’s most important responsibilities is the
recommendation of revisions to the FBI’s Quality Assurance Standards
(QAS) for DNA Analysis.
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SWGDAM 出版物
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SWGDAM法医DNA分析方法验证指导
3.1 Genetic Markers
3.6 Case Type Samples
3.2 Species Specificity
3.7 Population Studies
3.3 Sensitivity
3.8 Mixture Studies
3.4 Stability
3.9 PCR Based Studies
3.5. Precision & Accuracy
SWGDAM Validation Guidelines for Forensic DNA Analysis
Methods approved in November 2012 www.swgdam.org
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华夏白金物种特异性 – 非灵长类
200 rfu
Bovine
Chicken
•
•
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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.
700 rfu
Pig
Dog
Rabbit
Horse
Rat
Microbes
Sheep
Mouse
NTC
200 rfu
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),
horse, mouse and pig.
华夏白金一次性成功通过率
SWGDAM 3.4 The ability to obtain results from DNA
recovered from biological samples deposited on various
substrates and subjected to various environmental and
chemical insults should be evaluated.
• All sample types have ≥90% 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.
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样本准在3500xl上的准确度
SWGDAM 3.5 Precision
and accuracy of the assay
should be demonstrated.
Accuracy is the degree of
conformity of a measured
quantity to its actual (true)
value.
Size deviation of 42 population samples showed deviation <0.50bp for all alleles
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遗传标记在不同种族的人群中的分布
Allele for
D6S1043 Chinese
Sample#
1476
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0.0014
8
0.0047
9
0.0014
10
0.0332
11
0.1355
12
0.1314
12.3
0.0007
13
0.1216
14
0.1240
15
0.0152
16
0.0027
17
0.0335
17.3
0.0010
18
0.1762
18.2
0.0003
19
0.1535
20
0.0512
20.3
0.0007
21
0.0081
21.3
0.0027
22
0.0003
22.3
0.0003
RMP
0.0294
Hui
174
0.0086
0.0057
0.0402
Uyghur
205
0.0024
0.0024
Tibet
200
0.0275
0.0050
0.0575
0.1725
0.1375
Southern Northern
Han
Han
221
203
0.0023
0.0249
0.0973
0.1199
0.0045
0.1222
0.1425
0.0136
0.0045
0.0407
0.0045
0.1606
0.1207
0.1351
0.0220
0.2244
0.1488
0.1092
0.1351
0.0115
0.1098
0.0976
0.0171
0.0230
0.0366
0.1175
0.1050
0.0075
0.0025
0.0250
0.2069
0.1341
0.2200
0.1379
0.0517
0.1488
0.0415
0.1968
0.0656
0.0086
0.0057
0.0122
0.0825
0.0225
0.0025
0.0050
0.0100
0.0320
0.0309
SWGDAM 3.7 The distribution of
genetic markers in populations
should be determined in relevant
population groups.
0.0320
0.0911
0.1429
0.1576
0.1355
0.0123
0.0074
0.0271
0.1773
0.1601
0.0468
0.0074
0.0025
0.0024
0.0306
0.0341
0.0324
• Allele frequency for major Chinese population groups was studied.
• Data of D6S1043 was presented above, and the data with other markers was
published in UG.
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反应预混物保护带研究
Individual components
Component concentrations
• The Huaxia Platinum MM is robust to raw material change
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SWGDAM 3.9 The reaction
conditions needed to
provide the required degree
of specificity and
robustness should be
determined.
• 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.
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 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-Chromosome 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.
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提高Y-特异性的工作
• Primer mix
• Design/screen primer pairs with minimal female crossreactivity
• Master mix
• Lower the Mg and Taq enzyme concentrations to minimize
female cross-reactivity
• Multiple rounds of DOE were performed to optimize the
MM components
• PCR condition
• Increase the annealing/extension temperature to increase
male specificity
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Yfiler Plus 在男女混合DNA的表现
Internal Product Spec: The kit will recover full profile with average 40% ICB when amplified with
male:female mixtures of 1:1000
• Yfiler Plus recovered full profile with 1:1000 male:female mixture
• No extra peaks with Yfiler Plus, while multiple extra peaks with PPY23 and Yfiler
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Yfiler Plus高浓度女性DNA下的基线
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.
200rfu
68 bp
406 bp
Blue 271
Outside the
read region
Red 139/144
Red grass
Low female cross-reactivity (<175 PAT) within the read region are all
documented in the UG.
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3 微克女性DNA在三个盒子的比较
PAT
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.
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新的法医DNA标准 – ISO 18385
Manufacturing Quality Commitment
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
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