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GE Healthcare Life Sciences

Application Note 28-9905-56 AA OmniSwab

The use of quantitative PCR (qPCR) and DNA profiling to demonstrate the absence of contaminating human genomic DNA on OmniSwab
In this study, we have used statistical sampling and recognized processing methods to demonstrate that OmniSwab has undetectable levels of contaminating human genomic DNA. We processed OmniSwabs from three separate batches using two widely recognized DNA extraction methods (1,2). DNA levels were assessed by qPCR (Quantifiler Human DNA Quantification Kit, Applied Biosystems) and short tandem repeat (STR) DNA profiling (AmpFLSTR SGM Plus PCR Amplification Kit, Applied Biosystems).

Introduction
The Whatman OmniSwab is a sterile, buccal cell collection swab used in forensic databasing and criminal reference sample collection. The OmniSwab includes an ejectable brush-like swab head that enables efficient oral sampling and facilitates DNA extraction directly from the pad. Whatman Omniswabs are individually packaged in a clean-room environment and sterilized by gamma irradiation. Concern about contamination of disposable plasticware and reagents, during their manufacture, has led to the forensic community considering setting a regulatory standard for sample collection products and other consumables (3). Consequently, there is a need for information on the current levels of contaminating DNA that may be present on sampling products.

Methods
Study construction and DNA extraction Standard statistical sampling methods (MIL-STD-1916 Verification Level VII) (6) were used as a basis for the study. OmniSwabs (113 per batch) from three separate batches (batch size: 100,000) were randomly selected and processed using two standard DNA extraction methods. A DNA extraction procedure was carried out using a QIAamp DNA and Blood Mini kit (Qiagen) (2) via the buccal swab vacuum protocol according to manufacturers instructions or using Chelex-100 (1). Extraction-negative (no OmniSwab control), extraction-positive (OmniSwab-spiked with known donor gDNA [550 ng]), and process controls (Stock gDNA [550 ng] without OmniSwab) were included at routine intervals (every 24 samples) to ensure quality.

Results
Table 1a. QIAamp and Chelex extraction Quantifiler data for OmniSwab extracts using two extraction methods.
OmniSwab Lot No. OmniSwabs tested per extraction method QIAamp DNA and Blood Mini H039 G149 J209 113 113 113 Chelex-100 30 30 30 Quantifiler results per OmniSwab Not detected 143 143 143 >0.0 ng/l 0 0 0

Table 1b. DNA extraction results for QIAamp OmniSwab extracts (mean values for 12 sets of standards, n=36 per standard); similar results observed with Chelex 100 samples (data not shown).
Quantifiler DNA quantitation Acceptance criteria Standard curve Slope R2 IPC (Internal PCR control) Positive controls kit Negative control (NTC) -2.9 to -3.3 0.99 20-30 Cts DNA quantitated No DNA detected Pass specification Mean -3.188 0.997 27.239 Test result STD 0.043 0.002 0.354 Pass Pass Pass Pass Pass

DNA quantitation
Duplicate OmniSwab sample extracts (2 l) were quantitated using the Applied Biosystems Quantifiler Human DNA Quantification Kit on an Applied Biosystems 7900HT instrument according to manufacturers instructions (40 cycles). Each 96-well plate contained eight DNA standards in triplicate and two notemplate controls. Data analysis was carried out using the Applied Biosystems SDS 2.3 software (Ct threshold=0.2, baseline; start 3 cycle, Stop 15 cycle).

STR analysis
QIAamp and Chelex 100 DNA-extracts (n=29 per batch) were randomly selected from each of the three OmniSwab batches for amplification with AmpFLSTR SGM Plus kit (Applied Biosystems). PCR amplification reactions were carried out by adding 20 l sample extract to 30 l SGM Plus reaction mix. Thermal cycling was performed using an ABI GeneAmp PCR System 9700 following manufacturers thermal cycling conditions (28 cycles). The Applied Biosystems 3130XL Capillary Electrophoresis Genetic Analyzer was used to analyze each sample with standard and increased sensitivity methods. Samples (1 l) were denatured with HiDi formamide (10 l) and injected using injection time, 10 s; voltage, 3 kV (standard conditions) and injection time, 30 s; voltage, 4 kV (increased sensitivity conditions) (5). Data analysis was performed using GeneMapper 3.2 software (Applied Biosystems), with 50 RFU set as the threshold for allelic height peak calling.

Table 2. STR analysis - SGM Plus STR profiling 3130XL Capillary Electrophoresis (CE) Genetic Analyzer results (AmpFLSTR SGM Plus). All SGM Plus quality specifications were met. Zero alleles detected above background for all 174 OmniSwab extracts analyzed.
SGM Plus STR analysis Acceptance criteria Allele definition Contaminant definition Pass specification All peaks >50 RFU considered potential alleles Three or more peaks (>50 RFU), that cannot be explained as pull-up or stutter peaks at two or more loci, with the same profile present in a repeat sample Peak height >200 RFU Peak height ratio within 50% 11 loci called correctly 11 loci called correctly No allele (peak) present above background Pass Pass Pass Pass Pass Test results 0 alleles detected

Positive control homozygote alleles Positive control heterozygote alleles All loci called for kit positive control All loci called for known profile control Negative control

Table 3. SGM Plus STR analysis (50 RFU threshold setting) of OmniSwab extracts purified using two different extraction methods. FP (Full Profile) NP (No Profile).
Extraction method STR PCR cycle no. 28 28 28 Chelex 100 28 Injection time/ voltage 10 sec/ 3 kV 10 sec/ 3 kV 30 sec/ 4 kV 10 sec/ 3 kV Post PCR clean up None Min Elute Min Elute None Kit pos. control Kit neg. control Process control OmniSwab lot no. (n=29/lot) #H039 FP FP FP FP NP NP NP NP FP FP FP FP NP NP NP NP #G149 NP NP NP NP #J209 NP NP NP NP

Acceptance criteria
The specifications used in this study to determine acceptance were set using manufacturer kit specifications, in-house specifications and current literature (4). A contamination event was defined as three or more peaks, that cannot be explained as pull-up or stutter peaks at two or more loci, with the same profile present in a repeat sample.

QIAamp DNA Mini and Blood (buccal protocol)

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Fig 1. Typical profiles obtained using a 3130XL Genetic Analyzer. SGM Plus reaction profiles for process control (A) and SGM Plus positive kit control (B).

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Fig 2. Typical profiles obtained using SGM Plus. Reactions were processed using standard CE conditions pre (1) and post-PCR cleanup (2) and using higher sensitivity running conditions (3).

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DNA quantitation
Quantifiler analysis of serial gDNA dilutions demonstrated a minimal DNA detection of 1-10 pg/l. All experiments reliably met the kit sensitivity detection limit (23 pg/l). 100% of OmniSwabs (429) extracted using QIAamp (339) and Chelex 100 (90) gave zero Quantifiler results (Table 1a). No PCR inhibition was observed in any extract tested. All controls met manufacturers specifications (Table 1b). Based on statistical sampling of 113 samples tested from three different batches, it can be estimated that no more than 4 of the remaining 100,000 untested OmniSwabs in each batch are statistically likely to be nonconforming units (i.e., one that produces a nonzero Quantifiler result), at a 95% probability.

STR analysis
SGM Plus STR analysis of sample extracts purified using QIAamp DNA and Blood Mini (buccal protocol) and Chelex 100 (Table 3) demonstrated no detectable peaks above background that could not be explained as technology related artifacts (4). All controls met STR quality specifications (Table 2). When higher sensitivity conditions were used to increase detection of any contaminating DNA, no allelic peaks were observed above background from any of the OmniSwab extracts analyzed (Fig 2, profiles 2 and 3).

References
1. Walsh, P. S. et al, Chelex 100 as a medium for simple extraction of DNA for PCR-based typing from forensic material. Biotechniques 10(4), 506-13 (1991). 2. QIAGEN, Inc. QIAamp DNA Mini Kit and QIAamp DNA Blood Mini Kit Handbook. (April 2010). 3. Gill, P. et al, Manufacturer contamination of disposable plasticware and other reagentsan agreed position statement by ENFSI, SWGDAM, and BSAG. Forensic Sci Int Genet 4(4), 269270 (2010). 4. Butler, J. M., Forensic DNA typing: biology, technology, and genetics of STR markers, Elsevier Academic Press, Burlington (2005). 5. Forster, L. et al, Direct comparison of post-28-cycle PCR purification and modified capillary electrophoresis methods with the 34-cycle low copy number (LCN) method for analysis of trace forensic DNA samples. Forensic Sci Int Genet 2(4), 318-328 (2008). 6. Department of Defense test method, Standard DOD preferred methods for acceptance of product. MIL-STD 1916 (April 1996).

Conclusions
OmniSwab samples extracted using QIAamp and Chelex 100 (429 extractions in total) gave zero DNA quantitation results as determined by qPCR with Quantifiler. SGM Plus STR analysis of OmniSwab extracts (174 samples) gave no unexplained allelic peaks above background levels when a 50 RFU threshold setting was used. Additionally no unexpected contaminating allelic peaks were observed when sample extracts were reanalyzed with high-sensitivity analysis conditions (5) or after concentration by post-PCR cleanup.

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28-9905-56 AA 02/2011