Demonstrating the Efficacy of Ethylene Oxide Decontamination for the Reduction of DNA in Consumables used for DNA Extraction

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  • 1. Demonstrating the Efficacy of Ethylene Oxide Decontamination for the Reduction of DNA in Consumables used for DNA Extraction Ryo Hasegawa, Lisa Calandro, John Dektar, Harmandeep Gill, Yingjie Liu, Liwei Qi Life Technologies: 850 Lincoln Centre Dr. Foster City, CA 94404, USAINTRODUCTION Samples Treatment Efficacy Observations We spiked applicable samples with extracted DNA and cellular material (white Ethylene Significant and/or elimination depending on No discoloration or physical changesMinimizing human DNA contamination in the manufacture of forensic blood cells) to mimic conditions of contamination (Table 2). A lower input value was Oxide cyclesconsumables is a global challenge1. Recently, organizations throughout the chosen during the verification study to align the magnitude of the possible Physical changes where 100% Significant and/or elimination depending onworld including Standards Australia2 and the British Standards Institute3 contaminant. For the EO treatment, samples were placed in Tyvek® pouches (SPS Autoclaving time leakage with PrepFiler LySep™ columnshave introduced standards to address this issue as the increasing sensitivity Medical) which allowed gas to penetrate while blocking introduction ofof DNA analysis methods have placed the burden on manufacturers to contaminates. Ultraviolet Significant reduction depending on time Requires accessible surface and irradiation results in color change (“yellowing”)ensure products are free of extraneous DNA (Figures 1 & 2). Feasibility Study Verification Study N=5 N=7 Electron Improved Sensitivity of HID STR Kits beam radiation Identifiler® Plus Identifiler® Plus 1.5 mL PrepFiler® PrepFiler® Ineffective with Less than 4-fold reduction Results in color change (‘yellowing”) Identifiler® Samples 28 Cycles 29 Cycles Microcentrifuge Tube LySep Column LySep Column Gamma irradiation 50 ng and 500 ng 5 ng and 50 ng 31.25 pg White Blood Cells - (DNA equivalence) (DNA equivalence) Table 4. A summary of results from the feasibility study. 62.5 pg Extracted DNA - 50 ng and 500 ng 5 ng and 50 ng The high efficacy of dual cycle EO was reconfirmed during the verification study. No amplifiable DNA was detected with 5ng and 50ng of extracted 125 pg Table 2. A summary of prepared to evaluate the DNA decontamination efficacy. DNA (Figures 7 & 8). A minimum of 99.6% and 98.6% efficiency (based on A set of forensic samples was tested to evaluate the functional performance of the allele peak heights) was achieved with cellular materials spiked at 5ng and 250 pg post-treated plastics (Table 3). A DNA storage stability study was conducted to 50ng equivalent DNA respectively (Figures 7, 9 & 10). 500 pg ensure the extracts prepared and stored with the treated plastics were stable over EO Efficacy Extracted DNA EO Efficacy Cellular Material time (Table 3). PRE PRE 1 ng Quantifiler Human Quantifiler Human Amplification Results POST Amplification Results POSTGreen = Peak Height > 250rfu; Yellow = Peak Height <250 rfu; Red = Allelic Dropout at 50 rfu Functional Performance Study Replicates DNA Storage Stability Study 5ng 5ng 50ng 50ng 5ng 5ng 50ng 50ngFigure 1. An example of the increasing sensitivity of HID STR kits as more alleles are 40 40detected with improved chemistry. 10uL of epithelia cell suspension 12 Temp Time Points 38 38 36 36 DNA Extract from 0.02uL of Saliva 2.5 pg of Extracted DNA Ct Ct 0.2uL of liquid blood on cotton cloth 12 2, 4 and 11 34 34 4 C days 32 32 1uL of semen on cotton cloth 12 30 30 2 and 4 -20 C 28 28 weeks 0.2uL on liquid blood on denim 12 Figure 7. Quantification results from pre- and post-dual cycle EO treated samples (N=7). 0.167, 1.0 and 5.0 uL of liquid blood Serial liquid blood series (5, 1, 0.25, 0.1 and 0.025uL) 4 Replicates = 4 DNA Profile of Pre- and Post-EO Treated Extracted DNA 5mg of tooth 18 Pre-EO 5ng Pre-EO 50ng Avg. RFU Avg. RFU 5.6x Dilution 2067 3556 Table 3. A set of forensic samples tested during the functional performance study between pre- and post-treated plastics. Measurement of Efficacy Figure 2. Electropherogram from DNA extracted from 0.02uL of saliva (left) and 2.5pg of The efficacy of each treatment was evaluated by comparing the DNA yield of pre- Post-EO Post-EO extracted DNA (right). One and two alleles were detected above 50 RFU respectively. Samples were amplified with Identifiler Plus® for 29 cycles. and post-treated samples with Quantifiler® Human DNA Quantification Kit. The 50 RFU Threshold 50 RFU Threshold samples were extracted with either AutoMate Express™ or PrepFiler® AutomatedContaminants in forensic consumables can lead to severe consequences. systems. If a significant reduction in amplifiable DNA was detected (averageFor example, the “Phantom of Heilbronn” was an individual sought in Cτ≥35) , the effect of treatment on DNA profile was characterized with AmpFℓSTR®numerous cases for 15 years. Trace DNA profiles from a female individual Identifiler® Plus kit at 29 cycles (Figure 4). Figure 8. Electropherogram overlay (N=7) of pre- and post-dual cycle EO treated sampleswere detected at more than 40 crime scenes in Germany, Austria and spiked with 5ng (left) and 50ng (right) extracted DNA on the PrepFiler® LySep Column.France. The case was finally closed in 2009 by connecting the profiles to DNA/Forensic Materials Samples DNA Profile of Pre- and Post-EO Treated Cellular Materialcollection swabs contaminated by a production line worker. PrepFiler® LySep Non-Stick Microfuge Column Tubes (1.5 mL) 5ng 50ng Treatment Avg. RFU Pre-EO Pre-EOThe risks for extraneous DNA in collection devices and system 7x Dilution 1626 Avg. RFU 4140consumables can be significantly minimized. A number of measures have Ct = 28.9 DNA Extractionbeen suggested as standard practices at the manufacturing site2,3,4 (Figure3). A crucial step is applying a post-packaging decontamination process. PrepFiler® AutoMate Avg. RFU Post-EOThis will ensure sporadic events of contamination are effectively removed to Automated Express™ DNA Quantification 206 Ct = 35.1 Post-EOa level undetectable even under continual QC checks. 7500 Real-Time PCR System 50 RFU Threshold Post-EO Practices to Minimize Human DNA Contamination Identifiler® Plus STR Amplification Ct = 40.0 50 RFU Threshold Production Post-Production GeneAmp® Identifiler® PRC System 9700 Profile Evaluation Figure 9. Electropherogram overlay (N=7) of pre- and post-dual cycle EO treated samples Extensive Personal spiked with 5ng (left) of equivalent DNA on the PrepFiler® LySep Column. The 50ng sample Automation 3130xl Genetic AnalyzerProtective Equipment resulted in 4/7 non-amplifiable DNA and 3/7 (right) contained amplifiable DNA. Product Quality Treatment Figure 4. Experimental workflow utilized to analyze the DNA decontamination efficacy. Clean Production Quality Control Check Number of Alleles Detected with Post-EO Treated Cellular Material Expected Alleles Detected by Data Analysis Threshold Expected Alleles Detected from Post-EO Treated Sample Environment Control Checks Measurement of Functional Performance 50 5ng Eq. DNA of White Blood Cell 50 RFU 5ng Eq. DNA of White Blood Cell 15 RFU Threshold 15 RFU Threshold 50 RFU Threshold 50 RFU Threshold Minimize introduction of DNA Eliminate DNA Check for DNA The functional performance of the treated PrepFiler Express plastics was assessed 40 Figure 3. A summary of key measures to minimize contamination at the manufacturing site. once a preferred decontamination method was selected. DNA was extracted from a set of forensic samples with pre- and post-treated plastics using the AutoMate 30 RFU Express™ workflow. The DNA yield and DNA profile were assessed withMATERIALS AND METHODS Quantifiler® Human DNA Quantification Kit and Identifiler® respectively. The 20 15 RFU 10 stability of the DNA extracts were monitored overtime using the same quantificationDecontamination Techniques and STR kits. 0 20/189 Alleles 1/189 AllelesWe have evaluated the efficacy of various decontamination methods (Table Figure 10. Number of alleles detected for post-dual cycle EO treated 5ng of DNA eq. cellular1) as assessed by Shaw et al5, Archer et al6 and Gefrides et al7. These EXPERIMENTAL RESULTS material (N=7). A comparison was examined between nominal (50 RFU) and minimal (15 RFU)include ethylene oxide (EO), autoclaving, ultraviolet irradiation, electron data analysis threshold.beam radiation and gamma irradiation. The dual cycle EO treatment provided the most encouraging results during the The dual cycle EO treatment showed no adverse affect on the plastics. feasibility study. Other treatments resulted in marginal efficacy and/or physical Equivalent functional performance and DNA storage stability were Treatment Description Parameters changes to the plastics (Figures 5 & 6 and Table 4). The residual amount of EO demonstrated between the pre- and post-treated plastics (not shown). was well below the ISO10993-7:2008 limits for these plastics. Ethylene A gaseous chemical which destroys microbes and 1 and 2 cycles Efficacy of DNA Decontamination with Extracted DNA Quant Human Ct DNA = 50 ng Eq. DNA of Pristine Quant Human Ct DNA = 500 ng Eq. DNA of White Blood Cells CONCLUSIONS Oxide DNA by alkylation 40.0 50ng 40.0 500ng 37.5 37.5 EO UV The dual cycle EO treatment of PrepFiler® Express™ Plastics effectively High pressure steam to coagulate and destroy 2 and 3 hours Autoclaving proteins. at 121 C (15 psi) 35.0 35.0 Autoclave decontaminates trace levels of extraneous DNA without detrimental effect to 32.5 32.5 Ebeam the quantity and quality of DNA extracted. Manufacturing improvements Ct Ct Gamma 30.0 30.0 Ultraviolet UV light at short wavelength commonly used for 2, 4 and 8 hours coupled with post-packaging decontamination can significantly reduce the irradiation microbial decontamination and nucleic acid disruption under 4000 µW/Cm² 27.5 27.5 risk of extraneous DNA contamination. 25.0 25.0 TX TX Electron A form of ionizing energy characterized by low 0x x-C x -O x-C x -O 0H 2H 4H 8H Gy Gy Gy Gy Gy Gy Gy 0x x-C x-O x-C x -O 0H 2H 4H 8H Gy Gy Gy Gy Gy kGy k Gy kGy beam radiation penetration and high dosage which acts by breaking DNA chains. 20, 40, 80 and 100 kGy 1 1 2 2 0k 20k 40k 60k 00k 1 0k 30k 1 1 2 2 0k 20k 4 0k 60k 00k 1 0 30 50 REFERENCES Figure 5. The mean quantification results from treated samples spiked with 50 and 500ng of extracted DNA on PrepFiler® LySep columns. “C” and “O” refers to opened and closed configurations of the 1. Sullivan K, Johnson P, Rowlands D, Allen H (2004) New developments and challenges in the use of the UK DNA Gamma Emission of high energy photons to cause disruption Database: addressing the issue of contaminated consumables. Forensic Sci Int 146S:S175-176 31 and 50 kGy columns respectively. irradiation ionization resulting in DNA damage. 2. Standards Australia (2012) Minimizing the risk of contamination in products used to collect and analyse biological material for forensic DNA purposes. AS 54813-2012 Efficacy ofCtDNA Decontamination with Cellular Material Quant Human DNA = 50 ng Eq. DNA of White Blood Cells Quant Human Ct DNA = 500 ng Eq. DNA of White Blood Cells 3. The British Standards Institution (2012) Specification for consumables used in the collection, preservation and processing Table 1. Description of the various decontamination process and parameters evaluated. of material for forensic analysis. PAS 377:2012 40.0 40.0 50ng 500ng 4. Gill P, Rowlands D, Tully G, Bastisch I, Staples T, Scott P (2009) Manufacturer contamination of disposable plastics-ware 37.5 37.5 EO and other reagents-An agreed position statement by ENFSI, SWGDAM and BSAG. Forensic Sci Int: Genetics 4:269-270Considerations for Decontamination Methods 35.0 35.0 UV Autoclave 5. Shaw K, Sesardić I, Bristol N, Ames C, Dagnall K, Ellis C, Whittaker F, Daniel B (2008) Comparison of the effects of sterilisation techniques on subsequent DNA profiling. Int J Legal Med 122:29-33The following considerations were made when evaluating the processes. 32.5 32.5 Ebeam 6. Archer E, Allen H, Hopwood A, Rowlands D (2010) Validation of a dual cycle ethylene oxide treatment technique to Ct Ct Gamma remove DNA from consumables used in forensic laboratories. Forensic Sc Int: Genetics 4:239-243 The safety of the method. 30.0 30.0 7. Gefrides L, Powell M, Donley M, Kahn R (2010) UV irradiation and autoclave treatment for elimination of contaminating The efficacy to reduce detectable amounts of DNA from samples spiked 27.5 27.5 DNA from laboratory consumables. Forensic Sci Int: Genetics 4:89-94 25.0 25.0 with extracted DNA and representative cellular material. © 2012 Life Technologies Corporation. All rights reserved. The trademarks mentioned herein TX TX 0x 1x 2x 0H 2H 4H 8H 0H 2H 3H Gy Gy Gy Gy Gy kGy k Gy kGy 0x 1x 2x 0H 2H 4H 8H 0H 2H 3H Gy Gy Gy Gy Gy kGy k Gy kGy The functional performance of the plastics after treatment. 0k 20 k 40k 60 k 00k 1 0 30 50 0k 20 k 40k 60k 00k 1 0 30 50 are the property of Life Technologies Corporation or their respective owners. Tyvek is a The suitability at the production-scale. registered trademark of E. I. du Pont de Nemours and Company. Figure 6. The mean quantification results from treated samples spiked with 50 and 500ng of equivalent DNA of cellular material in a 1.5 mL microcentrifuge. For Forensic or Paternity Use Only.