Validation of a Multiplexed System for Quantification of Human DNA and Human Male DNA and Detection of PCR Inhibitors in Biological Samples

Transcription

1 18 th International Symposium on Human Identification Validation of a Multiplexed System for Quantification of Human DNA and Human Male DNA and Detection of PCR Inhibitors in Biological Samples Maura Barbisin, Rixun Fang, Cristin E. O Shea, Pius M. Brzoska, Lisa M. Calandro, Manohar R. Furtado and Jaiprakash G. Shewale Applied Biosystems, 850 Lincoln Centre Drive, Foster City CA 94404, USA

2 Outline Introduction Assay specificity and sensitivity Performance with mixture samples Performance with inhibited and degraded samples Correlation to STR kit performance Casework samples Conclusions 2

3 Objectives Develop a multiplexed real-time PCR assay to: Quantify total human DNA and human male DNA Determine the ratio of human male and female DNA Detect PCR inhibitors Allow selection of appropriate AmpFlSTR amplification kit Predict success with downstream STR amplification 3

4 Quantifiler Duo Kit: A Guide to Sample Assessment DNA Extract IPC Ct Value Quantification Re-Purification Heavy Inhibition STR Analysis Mixture Ratio Autosomal STR Y-Filer kit MiniFiler kit Identifiler kit 4 Partial Inhibition

5 Comparison to Quantifiler Human and Y Kits DNA standard is pooled human male genomic DNA PCR Reaction Mix optimized for expanded multiplex performance Increased amplicon size Ability to obtain ratio of human and male DNA in a mixture sample in a single reaction Dual quantification of human and male DNA minimizes sample consumption Assay designed to guide selection of appropriate AmpFlSTR Kit chemistry Maximizes recovery of interpretable STR profiles 5

6 Configuration of Quantifiler Duo Kit Target Marker Size Dye Human DNA RPPH1 (Ribonuclease P RNA component H1) 140 bp VIC Human Male DNA SRY (Sex determining Region Y) 130 bp FAM IPC Artificial Template 130 bp NED Similar amplicon sizes to avoid preferential PCR. Validated on the 7500 Real Time PCR System using SDS v1.2.3 software. 6

7 Quantifiler Duo Kit Components Component Quantifiler Duo Primer Mix Quantifiler Duo PCR Reaction Mix Quantifiler Duo DNA Standard Quantifiler Duo Dilution Buffer Composition Primer pairs for amplification of RPPH1, SRY and IPC. The TaqMan probes for RPPH1, SRY and IPC are labeled with VIC, FAM and NED dyes, respectively. In addition, this reagent contains IPC template. AmpliTaq Gold DNA Polymerase in buffer and salts. Pooled human male genomic DNA 10 mm Tris HCl buffer, ph 8.0 containing 0.1 mm EDTA The structure of the Duo kit is very similar to the Quantifiler Human and Y kits 7

8 Species Specificity Tested RPPH1 SRY Species Average Ct Average Ct Orangutan Chimpanzee A Chimpanzee B Gorilla A Gorilla B Macaque Dog Cow Pig Cat Horse Sheep Chicken Fish Rabbit Mouse Rat Hamster Human Male Human Female E. coli Pseudomonas Neisseria Staphylococcus Saccharomyces Candida RPPH1 SRY 5 or 10 ng of DNA from the indicated species was tested. Human male and female Human male Chimp A & B Human female 8

9 Standard Curves and IPC C T Values Standard Curve Ave Ct SRY-FAM RPPH1-VIC 22 IPC-NED Log of Concentration 9

10 Sensitivity Study Sample Sample Source Expected Quantity Measured Quantity (RPPH1) Measured Quantity (SRY) ng/μl ng/μl ng/μl 1 Human Male Blood Human Male Blood Human Male Blood Human Male Blood Human Male Blood Human Male Blood Human Male Blood The last two dilutions are below the limit of quantification of the standard curve. Quantifiler Duo is capable of detecting as low as ~ 6 pg/µl of human DNA. Caution should be exercised in the interpretation of low DNA concentrations as quantities obtained with any PCR assay may not be accurate due to stochastic effects. 10

11 Analysis of Male & Female DNA Mixtures Mixture Study II SRY (FAM) RPPH1 (VIC) Quantity (ng/ul) :0 1:50 1:100 1:200 1:500 1:800 1:1000 0:1 Ratio (male: female) Male DNA added at a constant concentration of ng/µl 11

12 Analysis of Male & Female DNA Mixtures Male/Female SRYQuantity RPPH1 Quantity SRY/RPPH1 SRY/RPPH1 DNA ratio ng/ul ng/ul expected ratio measured ratio 1: :1 1:0.96 1: :51 1: : :101 1: : :201 1: : :501 1: : :801 1: : :1001 1: :1 Female :0 1:800 Yfiler kit profile ~0.25 ng DNA/rxn 1:1000 Male DNA added at a constant concentration of ng/µl 12

13 Hematin Inhibited Samples C T values Inhibitor: Hematin SRY (FAM) RPPH1 (VIC) IPC (NED) Ct uM 2.5uM 5uM 7.5uM 10uM 12.5uM 15uM 17.5uM 20uM 40uM The effect of the inhibitor on the C T values is similar for all three targets 13

14 Hematin Inhibited Samples Correlation with Identifiler profiles control full IPC Ct in hematin inhibited samples IPC Ct ctrl 2.5 µm 5 µm 7.5 µm 10 µm 12.5 µm 15 µm 17.5 µm 20 µm 40 µm 7.5uM 10uM 12.5uM full full Full-low peak height 15uM partial 40uM no profile 14

15 Degraded DNA Results with Degraded DNA Samples Concentration (ng/ul) Quantifiler Duo RPPH1 Quantifiler Duo SRY Intact 0.002U 0.01U 0.02U 0.05U 0.1U 0.2U DNA degradation State 15

16 Degraded DNA Comparison with Quantifiler Kits Results with Degraded DNA Samples Quant Results (ng/ul) Quantifiler Human Quantifiler Y Quantifiler Duo RPPH1 Quantifiler Duo SRY Intact 0.002U 0.01U 0.02U 0.05U 0.1U 0.2U DNA degradation State Quantification of degraded DNA was performed using Quantifiler Duo, Human, and Y kits. 16

17 Degraded DNA Identifiler and MiniFiler Profiles Identifiler MiniFiler 0 U DNase I U DNase I 0.01 U DNase I 0.02 U DNase I 0.05 U DNase I 0.1 U DNase I 0.2 U DNase I 17

18 Post-coital Samples Collected at Different Time Intervals Quantitation of post-coital samples (e-cell fraction) Quantitation of post-coital samples (sperm fraction) RPPH1 SRY RPPH1 Quantity (ng/ul) Quantity (ng/ul) SRY h 8 h 12 h 24 h h 8 h 12 h 24 h Quantification results were obtained after differential extraction 18

19 Post-coital Samples: E-cell Fraction Identifiler Kit Profile (1 ng DNA/rxn) E cell 4 h E cell 8 h E cell 12 h E cell 24 h rfu

20 Post-coital Samples: Sperm Fraction Identifiler Kit Profile (1 ng DNA/rxn) Sperm 4 h Sperm 8 h Sperm 12 h Sperm 24 h rfu

21 Post-coital Samples: Sperm Fraction Yfiler Kit Profile (1 ng DNA/rxn) Sperm 4 h Sperm 8 h Sperm 12 h Sperm 24 h rfu

22 More Case-type Samples Organic Extraction Sample # Sample Type Quantity (SRY) ng/µl Quantity (RPPH1) ng/µl Ct Value (IPC) SRY-RPPH1 % difference Blood stain on denim is a challenging sample, due to the inhibitors present on the denim substrate and in the blood, as shown by the IPC C T increase 22 1 Saliva Swab (a) Saliva Swab (b) Blood Stain on Fabric (a) Blood Stain on Fabric (b) Blood Stain on Denim Blood Stain on Filter Paper Blood Stain on Fabric spiked 7 with Inhibitor Mix Semen Stain on Fabric

23 Case-type Samples: Organic Extraction Identifiler and MiniFiler Profiles Identifiler Sample # MiniFiler

24 Casework Samples Verification Test Sites Site Samples Extraction Quant range (ng/μl) STR kits USA (1) 24 Organic Identifiler, Yfiler USA (2) 22 Qiagen Identifiler, Yfiler Europe 20 Invitrogen Identifiler, Yfiler Australia 20 Chelex (28.16) Profiler Plus /COfiler 24

25 Casework Samples USA (2): Swab of a Cell Phone Data kindly provided by San Diego Police Department SAMPLE TYPE ORIGIN SOURCE EXTRACTION RPPH1 SRY IPC C cell swab cell phone S (fem?) Qiagen Identifiler Yfiler 4000 rfu 4000 rfu 1000 rfu 1500 rfu The profiles confirm that the sample is a male/female mixture, as assessed by the Quantifiler Duo quantification results. 25

26 Casework Samples USA (2): Cigar Butt Data kindly provided by San Diego Police Department SAMPLE TYPE ORIGIN SOURCE EXTRACTION RPPH1 SRY IPC ACQ cigar cigar S (male) Qiagen Identifiler Yfiler 4000 rfu 6000 rfu The profiles confirm that this sample is from a male single individual, as assessed by the Quantifiler Duo quantification results 26

27 Casework Samples USA(2): Mixed Bloodstain Data kindly provided by San Diego Police Department SAMPLE TYPE ORIGIN SOURCE EXTRACTION RPPH1 SRY IPC SAM/PTO blood stain fabric(?) M/M Qiagen Identifiler Yfiler 4000 rfu 2000 rfu According to the profiles, the sample is a mixture of two male individuals. Quantifiler Duo cannot predict mixture of two male or two female individuals. 27

28 Casework Samples Europe: Sweat from a Used Latex Glove Data kindly provided by Carabinieri-RIS SAMPLE TYPE ORIGIN SOURCE EXTRACTION RPPH1 SRY IPC U sweat glove? Charge Switch Identifiler The profile confirms that there is no amplifiable DNA in the sample, as assessed by the Quantifiler Duo quantification results 500 rfu 28

29 Casework Samples Australia: Epithelial Cells from Shoes Data kindly provided by PathWest Laboratory Medicine WA SAMPLE TYPE ORIGIN SOURCE EXTRACTION RPPH1 SRY IPC FB1 Epithelials Shoes S Chelex Profiler Plus COfiler 2000 rfu 2000 rfu The profiles confirm that this sample is from a male single individual, as assessed by the Quantifiler Duo quantification results 29

30 Conclusions Quantifiler Duo is a forensic DNA sample assessment tool which provides information on: Quantity of human and male DNA Mixture ratio PCR inhibitors Single copy targets provide reliable quantification results The assay is specific for human and higher primate DNA High dynamic range of quantification from 23 pg/µl to 50 ng/µl; LOD is 6 pg/µl Quantities below this range require addition of the maximum volume allowed by the STR kit Quantification results obtained using the Quantifiler Duo Kit correlate well with Identifiler, Yfiler and MiniFiler Kit performance Realistic estimates of DNA concentration in compromised samples due to amplicon size Prediction of accurate DNA input amounts which result in high quality STR profiling results Selection of appropriate AmpFlSTR Kit to use downstream 30

31 Acknowledgements Verification Test Sites PathWest Laboratory Medicine WA (AU) Gavin Turbett, Merica Buxton, Douglas Jeffery Ministry of Justice-Netherlands Forensic Institute (NFI) (EU) Wiljo de Leeuw Centre of Forensic Sciences (Canada) Roger Frappier, Loretta D Costa San Diego Police Department (USA) Adam Dutra, Shawn Montpetit, Amy Rogala Carabinieri-Reparto Investigazioni Scientifiche (EU) Magg. Marco Pizzamiglio Other sites Applied Markets Research and Development Hemant Pawar, Max Brevnov, LinLin Chou Applied Markets Marketing Heidi Kijenski Applied Markets Product Development Lindy Kauffman, Yixin Li, Sabine Short, Beth Ladin 31

32 Legal Statements For Research, Forensic or Paternity Use Only. Not for use in diagnostic procedures. Purchase of the Quantifiler Duo Kit includes an immunity from suit under patents specified in the product insert to use only the amount purchased solely in forensic and paternity testing, and also for the purchaser s own internal research. No other patent rights are conveyed expressly, by implication, or by estoppel. For further information contact the Director of Licensing, Applied Biosystems, 850 Lincoln Centre Drive, Foster City, California 94404, USA. Not for re-sale. Applera, Applied Biosystems, AB (Design), AmpFLSTR, COfiler, Identifiler, Profiler Plus, Quantifiler, TaqMan, VIC, and Y-filer are registered trademarks and FAM, MiniFiler, and NED are trademarks of Applera Corporation or its subsidiaries in the US and/or certain other countries. AmpliTaq Gold is a registered trademark of Roche Molecular Systems, Inc. All other trademarks are the sole property of their respective owners. 32

33 Thank You Q & A