Influence of Amount of Starting Material for DNA Extraction on

Article pubs.acs.org/JAFC

Influence of Amount of Starting Material for DNA Extraction on Detection of Low-Level Presence of Genetically Engineered Traits∥ Tigst Demeke,*,† Anh Phan,‡ Indira Ratnayaka,† Michelle Holigroski,† and G. Ronald Jenkins§ †

Canadian Grain Commission, 1404-303 Main Street, Winnipeg, Manitoba R3C 3G8, Canada Corporate Services, Canadian Grain Commission, 303 Main Street, Winnipeg, Mantiboa R3C 3G8, Canada § Maple Woods College, 2601 NE Barry Road, Kansas City, Missouri 64156-1299, United States ‡

ABSTRACT: Two laboratories independently examined how the amount of starting material influences DNA extraction efficiency and, ultimately, the detection of low-level presence of genetically engineered (GE) traits in commercialized grains. GE traits from one maize, two canola, and two soybean samples were used as prototypical models in the study design as well as two commonly used DNA extraction methods, a small scale (0.1 and 0.2 g samples) and a large scale (1.0 and 2.0 g samples). The DNA samples were fortified (spiked) at 0.1 and 0.01% (w/w) levels. The amount of DNA recovery varied between the two laboratories, although a sufficient amount of DNA was obtained to perform replicate PCR analysis by both laboratories. Reliable detection of all five events was achieved by both laboratories at 0.1% level using either small-scale or large-scale DNA extractions. Reliable detection of the GE events was achieved at 0.01% level for soybean and canola but not for maize. Variability was observed among the two laboratories in terms of the Ct values generated. There was no difference between small-scale and large-scale DNA extraction methods for qualitative PCR detections of all five GE events. KEYWORDS: genetically engineered events, PCR, A2704, HCN92, OXY235, DP305423, MIR604, canola, soybean, maize

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INTRODUCTION As grains move through the production and distribution chain, comingling of similar types and qualities routinely occurs, making the low-level presence (formerly known as adventitious presence) of mixed varieties a reality in bulk grain handling systems. With the advances of modern agricultural biotechnology, complex challenges have been introduced into global markets when trading grains. These challenges have become self-evident between exporting countries that produce a surplus of genetically engineered (GE) agricultural products and importing countries that apply different authorization standards to the same commodities. Grain quality and product compliance measures, based on sound scientific principles and reasonable outcomes, are necessary for the purpose of facilitating marketing of GE grain products. Low-level presence of unapproved or discontinued GE materials in non-GE commercial grains, oilseeds, and foodstuffs has been a contentious regulatory issue that often affects international grain trade.1,2 A low-level presence of differing varieties potentially occurs when (1) naturally occurring drift of GE pollen, carried by wind and insects, contaminates neighboring fields; (2) planting equipment is shared between different types of farming operations; (3) equipment such as augers, conveyors, and elevators handle both GE and non-GE crops; (4) GE crops comingle with non-GE crops in grain bins and drying equipment; (5) harvesting machinery, such as combines, are utilized to harvest both GE and non-GE crops; and (6) during the field trial stage of development, GE seeds located in test plots infiltrate nearby plots being grown to generate foundation seed. Two examples of a low-level presence of unapproved GE events that significantly affected commercial grain trade include FP967 flax in Canada2 and LLRice601 in the United States.3 Many countries have developed legislation regarding tolerance © 2014 American Chemical Society

and traceability requirements for GE traits. However, there is a wide variation in tolerance and traceability requirements among countries, thus affecting grain trade.1 Some of the challenges for detection of trace levels of GE traits include sampling and sample preparation, the amount of starting material when DNA extraction is performed, the amount of DNA recovered during the extraction, and the integrity of the DNA used for PCR. According to European Commission Regulation No. 619/ 2011,4 reliable detection of GE traits can be achieved at a 0.1% level. For the detection of FP967 transgenic flax event, detection limits of 0.0035%2 and 0.001%5 have been reported. However, there are some controversial opinions regarding laboratories’ capacity to accurately and reliably detect the low-level presence of GE materials below the 0.1% level.6 One study in particular suggests that relatively large amounts of starting material for DNA extraction (e.g., 2.0 g) provide better reproducibility and precision in the final analytical result.7 Many of the commercially available DNA extraction kits cannot be used for extraction of DNA from relatively large amounts of ground samples such as 2.0 g (the kits are usually limited to ≤0.2 g ground material). The traditional sodium dodecyl sulfate (SDS)- and cetylmethylammonium bromide (CTAB)based methods can be used for extraction of DNA from relatively large amounts of samples such as 1.0−5.0 g. However, both SDS- and CTAB-based DNA extraction methods are timeconsuming and tedious. In addition, toxic chemicals such as phenol and chloroform are used for the large-scale DNA extraction methods. To date, the impact of the amount of starting Received: Revised: Accepted: Published: 4349

February 5, 2014 April 3, 2014 April 18, 2014 April 18, 2014 dx.doi.org/10.1021/jf500610w | J. Agric. Food Chem. 2014, 62, 4349−4358

Journal of Agricultural and Food Chemistry

Article

Canada). DNA samples used for a given PCR were quantified at the same time for all methods. The Abs260/280 and Abs260/230 ratios for the genomic DNA were determined with a SpectraMax Plus 384 (MDS Analytical Technologies, Sunnyvale, CA, USA) at the CGC. At the USDA/GIPSA, DNA was quantified using a fluorometric assay with a Luminometer 20/20 instrument (Turner Biosystems Inc., Sunnyvale, CA, USA) in conjunction with the Quant-iT PicoGreen (PG) assay kit (Molecular Probes). Stock DNA samples were diluted between 1:250 and 1:500 with 0.5× TE (Tris-EDTA) buffer to a target concentration of 20−200 pg/μL. The PG reagent was prepared according to the manufacturer’s protocol; the diluted stock DNA test and reference samples were mixed 1:1 with PG reagent to a final volume of 200 μL to produce 1:500 to 1:1000 final dilutions and compared with fluorometric measurements from a standard curve. A calibration curve was generated from λ phage DNA, supplied by the manufacturer (Molecular Probes) at a stock concentration of 100 ng/μL and diluted to 250, 125, 62.5, 31.3, and 0.0 pg/μL with 0.5× TE buffer. Extracted DNA test samples were diluted to a working stock concentration of 20 ng/μL. Polymerase Chain Reaction. For qualitative real-time PCR, the 25 μL volume contained 1× TaqMan Universal Master Mix, primers, probes, and 100 ng of genomic DNA. The DNA sequences and concentration of primers and probes used are provided in Table 1. Fortified reference DNA samples (0.01 and 0.1%), non-GE DNA, and no template controls were included for each PCR experiment. Both laboratories performed PCR in triplicate using an ABI 7500 real-time PCR system. The thermal profile for all events, except the OXY235 canola event consisted of initial holds for 2 min at 50 °C and for 6 min at 95 °C, followed by 45 cycles of 15 s at 95 °C and a 1 min annealing/ extension step at 60 °C. For the OXY235 canola event, the thermal profile consisted of initial holds for 2 min at 50 °C and for 10 min at 95 °C, followed by 50 cycles of 15 s at 95 °C and 1 min annealing/ extension step at 60 °C. Statistical Analysis of Data. The data set of Ct values from realtime PCR was analyzed using SAS software (version 9.2 SAS Institute Inc., Cary, NC, USA). For purposes of ignoring distinction between grain types (i.e., maize, soybean, and canola) used in the study, the potential sources of variance in observed Ct values in the combined data set were composed of (1) laboratory differences (CGC vs USDA), (2) extraction method (CTAB vs Fast ID), (3) fortification level (0.1 vs 0.01% GM), (4) starting amount of sample (0.1 and 0.2 g with Fast ID vs 1 and 2 g with CTAB-Zymo), and (5) replication (×3) of Ct measurements within each level of sample amount. As distribution-fitting based on the small number of observations (n= 6) relevant to comparisons of interest would not be robust, the Wilcoxon nonparametric test was used as it does not make assumptions about the distribution of observed Ct values.9 The Wilcoxon option was used with PROC NPAR1WAY10 for making relative comparison of Ct values between groups. In addition to P values for Wilcoxon one-sided and two-sided two-group comparisons, analysis outputs included chisquare probabilities for the Kruskall−Wallis test, which were used to assess statistical difference between groups, as group comparisons did not assume matched pairs for observed Ct values.

material for DNA extraction on the reliability of detection of low concentrations of GE traits by PCR has not been systematically studied. Therefore, this study was designed to compare the effect of using small-scale (0.1 and 0.2 g of starting materials) and large-scale (1.0 and 2.0 g of starting materials) DNA extractions on the detection of the low-level presence of GE traits (0.01 and 0.1% fortified or spiked samples) using data collected from two independent laboratories. It was also aimed to determine if there was variability among different crops and GE events in terms of detection of the low-level presence of GE traits by performing statistical analysis to evaluate the results.

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MATERIALS AND METHODS

Seed Sources and Sample Preparation. Seeds of Armor BX GE canola variety (event OXY235) and Innovator (event HCN92) were received from the Oilseeds Monitoring unit of the Canadian Grain Commission (CGC). Certified seeds of non-GE canola variety, Eagle, were purchased from the American Oil Chemists’ Society (AOCS, Urbana, IL, USA). The OXY235 and HCN92 GE canola events were not detected in the non-GE variety. For the DP305423 soybean event, certified ground reference materials ERM-BF426C (1%) and ERMBF426D (10%) were used for making the 0.01 and 0.1% spike samples with a non-GE soybean variety. For the A2704-12 soybean event, certified seeds of the GE soybean variety PS2295 LL (Bayer Crop Science) were used for fortification. Seeds of the non-GE soybean variety Colby, obtained from Thompsons Ltd. (Chatham, ON, Canada), were used for fortification for both soybean events. The GE events DP305423 and A2704-12 were not detected in the Colby soybean variety (data not shown). Certified heterozygous maize reference samples 0407A (non-GE variety, Syngenta) and 0607A2 (MIR604eVent, Syngenta) were purchased from AOCS and used for fortification at 0.01 and 0.1% levels. The canola and soybean samples (with the exception of DP305423 ground certified reference material) were ground with a Retsch model ZM100 Centrifugal Grinding Mill (Fischer Scientific, Ottawa, ON, Canada) using a 1.0 mm sieve. For 0.01% GE gravimetrically fortified samples, 0.01 g of GE powder sample was mixed with 99.99 g of nonGE powder sample, whereas for 0.1% fortified GE sample, 0.1 g of GE powder sample was mixed with 99.90 g of non-GE powder sample. The fortified powder samples were placed in 500 mL autoclaved Nalgene centrifuge tubes (Thermo Fisher Scientific, Ottawa, ON, Canada) and thoroughly mixed manually to maintain homogeneity. Subsamples of the ground materials from the same source were used by USDA/GIPSA and CGC laboratories for DNA extraction and PCR. DNA Extraction and Quantification. For extraction of DNA from 0.1 and 0.2 g samples, a Fast ID Genomic DNA Extraction Kit (Genetic ID, Fairfield, IA, USA) was used following the manufacturer’s recommendations. For soybean and maize, the procedure was slightly modified at CGC as the supernatant was not sufficient for the followup steps. A single 0.25 in. ceramic grinding ball (Qbiogen, Carlsbad, CA, USA) was added to each 2.0 mL centrifuge tube containing the sample, plus 1400 μL of lysis buffer, and the centrifuge tubes were pulverized in a mixer mill (Qiagen TissueLyser II, Toronto, ON, Canada) at 30 Hz for 60 s before incubation. For extraction of DNA from 1 and 2 g samples, the CTAB DNA extraction method was followed.8 Ten milliliters of extraction buffer was used for both 1 and 2 g samples. Before PCR assay, the DNA extracted with the CTAB-based method was purified with the DNA Clean & Concentrator 25 kit (Zymo Research, Cedarlane Laboratories, Ltd., Burlington, ON, Canada; and Zymo Research Corp., Irvine, CA, USA, for CGC and USDA/GIPSA laboratories, respectively). DNA was quantified by fluorometry with a PicoGreen Assay Kit (Molecular Probes, Eugene, OR, USA) as recommended by the manufacturer. Assays were performed in 96-well fluorescence microtiter plates (Thermo Scientific, Waltham, MA, USA), and fluorescence was measured on a SpectraMax GeminiXS (MDS Analytical Technologies, formerly known as Molecular Devices, Toronto, ON,

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RESULTS AND DISCUSSION DNA Yield and Quality. DNA was extracted from finely ground canola, soybean, and maize samples to determine whether adequate amounts of intact DNA could be recovered and used to detect a low-level presence of GE traits by PCR. Highly variable DNA yields were obtained and verified by both the CGC and USDA/GIPSA laboratories for the Fast ID and CTAB-based DNA extraction methods (Table 2). CGC generally recovered greater amounts of DNA that ranged from 1.6 ± 0.3 to 182.9 ± 2.2 μg, whereas USDA/GIPSA generally recovered lesser amounts of DNA that ranged from 1.1 ± 0.2 to 84.0 ± 5.8 μg. Additionally, the data revealed that in most cases the Fast ID kit provided greater quantities of recovered DNA when using larger amounts of finely ground starting material 4350

dx.doi.org/10.1021/jf500610w | J. Agric. Food Chem. 2014, 62, 4349−4358


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Table 1. Primer and Probe DNA Sequences and Concentrations Used for Qualitative Real-Time PCR Assays primer name MDB685-F KVM180-R TM 029 Taqman probe OXYRG OXYRV OXYRP- probe MDB510-F MDB 511-R TM 003 Taqman Probe DP-305-F1 DP-305-R5 DP-305 probe KVM175 SMO001 TM031 TaqMan probe Lec for2 GMO3-126 Rev Lec TaqMan probe MIR604 primer F MIR604 primer R MIR604 TaqMan probe Zm Adh1 primer F Zm Adh1 primer R Zm Adh1 TaqMan probe

crop and event

sequence (5′−3′)

primer/probe concn (nM)

amplicon size (bp)

ref

400 400 200 300 300 150 200 200 200 800 500 220 400 400 200 550 550 100 600 300 200 300 300 200

95

19

124

20

101

19

93

21

64

22

74

21

76

23

135

23

canola (HCN92)

GTT GCG GTT CTG TCA GTT CC CGA CCG GCG CTG ATA TAT GA FAM-TCC CGC GTC ATC GGC GG-TAMRA canola (OXY235) GAT AGA TGG TGG TGT GAG TCT TGT CCT AAC TTT TGG TGT GAT GAT GCT 6-FAM-TGC CAT CAG CTG ACA CGC CGT GC-TAMRA canola (CruA) GGC CAG GGT TTC CGT GAT CCG TCG TTG TAG AAC CAT TGG VIC-AGT CCT TAT GTG CTC CAC TTT CTG GTG CA 3′-TAMRA soybean (DP305423) CGT GTT CTC TTT TTG GCT AGC GTG ACC AAT GAA TAC ATA ACA CAA ACT A 6-FAM-TGA CAC AAA TGA TTT TCA TAC AAA AGT CGA GA-TAMRA soybean (A2704-12) GCA AAA AAG CGG TTA GCT CCT ATT CAG GCT GCG CAA CTG TT 6-FAM-CGG TCC TCC GAT CGC CCT TCC-TAMRA soybean (lectin) CCA GCT TCG CCG CTT CCT TC GAA GGC AAG CCC ATC TGC AAG CC 6-FAM-CTT CAC CTT CTA TGC CCC TGA CAC-TAMRA maize (MIR604) GCG CAC GCA ATT CAA CAG GGT CAT AAC GTG ACT CCC TTA ATT CT FAM-AGG CGG GAA ACG ACA ATC TGA TCA TG-TAMRA maize (Adh1) CGT CGT TTC CCA TCT CTT CCT CC CCA CTC CGA GAC CCT CAG TC VIC-AAT CAG GGC TCA TTT TCT CGC TCC TCA-TAMRA

Table 2. Summary of Average DNA Yield for Fast ID and CTAB-Based DNA Extraction Methodsa average DNA yield (μg) DNA extraction trait

0.01% CGC

0.1%

amount (g)

method

OXY235 canola

0.1 0.2 1.0 2.0

Fast ID Fast ID CTAB CTAB

3.9 8.2 81.3 182.9

± ± ± ±

1.1 3.1 9.4 2.2

1.3 1.9 13.1 9.1

GIPSA ± ± ± ±

0.0 0.1 1.4 2.2

3.2 9.0 104.5 164.9

CGC ± ± ± ±

0.7 2.2 8.0 17.6

1.1 1.6 12.9 8.6

GIPSA ± ± ± ±

0.2 0.1 1.6 3.4

HCN92 canola

0.1 0.2 1.0 2.0


3.1 5.9 48.9 166.9

± ± ± ±

0.9 1.8 1.3 19.7

1.5 2.3 8.6 10.8

± ± ± ±

0.1 0.3 1.0 0.9

1.6 2.0 55.6 170.6

± ± ± ±

0.3 0.1 15.4 6.8

1.4 1.7 7.2 10.1

± ± ± ±

0.0 0.1 2.6 0.7

DP305423 soybean

0.1 0.2 1.0 2.0


5.2 6.2 75.5 90.3

± ± ± ±

0.3 0.5 5.9 10

2.1 3.5 12.6 85.5

± ± ± ±

0.6 0.4 1.8 4.2

6.0 4.1 54.6 76.8

± ± ± ±

2.1 0.4 29.5 16.5

1.8 3.4 13.5 74.0

± ± ± ±

0.2 0.3 2.5 6.9

A2704-12 soybean

0.1 0.2 1.0 2.0


9.1 9.3 49.4 54.9

± ± ± ±

0.2 1.7 3.3 25.3

2.0 3.1 14.1 70.2

± ± ± ±

0.6 1.2 1.5 23.4

10.2 7.7 64.9 73.0

± ± ± ±

1.6 0.9 0.4 1.4

1.6 3.1 14.2 84.0

± ± ± ±

0.2 0.3 0.5 5.8

MIR604 maize

0.1 0.2 1.0 2.0


3.9 7.8 24.2 56.5

± ± ± ±

0.8 3.3 1.4 0.4

2.7 4.2 18.2 48.1

± ± ± ±

0.4 0.5 1.6 3.4

4.6 4.0 24.5 58.3

± ± ± ±

1.6 2.7 0.7 3.3

3.0 4.4 18.6 50.2

± ± ± ±

0.2 0.5 0.9 3.8

The ± values are average of two DNA extractions. Fast ID, Fast ID DNA extraction method; CTAB, CTAB-based DNA extraction method (before Zymo purification of the DNA).

a

(Fast ID comparison of 0.1 vs 0.2 g). Similar observations were seen with the CTAB extraction method (comparison of 1.0 vs 2.0 g). Atypical quantities of recovered DNA were generated by

CGC on the two soybean samples and the single maize sample using Fast ID kits. For CGC, consistently higher canola, soybean, and maize DNA yields were obtained for the Fast ID method 4351



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compared with USDA/GIPSA with the exception of 0.2 g maize sample fortified at 0.1%. The likely explanation for this observation could be that the CGC extraction method included a ceramic grinding ball in the centrifuge tube containing buffer and pulverization with the Mixer Mill for soybean and maize, which created a finer particle size thereby leading to a higher extraction efficiency (see Materials and Methods). With the Fast ID DNA extraction method, the DNA yield ranged from 1.1 ± 0.2 to 10.2 ± 1.6 μg. Six hundred nanograms of DNA was sufficient to carry out the experiments in triplicate for both the transgene and the endogenous control. As predicted, much higher yields of DNA were obtained with the large scale CTAB-based extraction method (1.0/2.0 g sample) compared with the small-scale Fast ID extraction method (0.1/0.2 g sample (Table 2)). Interestingly, there were large differences in DNA yields between CGC and USDA/ GIPSA laboratories using the CTAB extraction method. The CTAB protocol used by the two laboratories had some differences that might explain these observations. The USDA/ GIPSA protocol excluded the reducing agent, β-mercaptoethanol, with their CTAB method during steps that required chloroform/ isoamyl alcohol extractions. For the CGC, β-mercaptoethanol (0.01 M), phenol/chloroform/isoamyl alcohol 25:24:1 (1×) and chloroform/isoamyl alcohol 24:1 (2×) were used in the extraction steps. Modification of the DNA extraction protocol may have partially contributed to the variation in total canola DNA yield, even though the soybean and maize DNA yields were not affected as much. Because DNA yield extracted from the same amount of starting material provided significant variability, the use of equivalent amounts of DNA in the PCR was critical.

Table 3. Abs260/280 and Abs260/230 Ratios (CGC Laboratory) for Samples Fortified with GE Traits Obtained from Two DNA Extraction Methods average absorbance ratios for DNA extraction method

fortification level (%)

OXY235 canola

Fast ID CTAB Fast ID CTAB

0.01 0.01 0.1 0.1

1.77 1.87 1.75 1.93

0.55 2.03 0.60 2.39

HCN92 canola


0.01 0.01 0.1 0.1

1.73 1.77 1.47 1.92

0.27 1.64 0.48 1.83

A2704 soybean


0.01 0.01 0.1 0.1

1.90 1.90 1.88 1.90

1.20 2.20 1.15 2.00

DP305423 soybean


0.01 0.01 0.1 0.1

1.84 1.93 1.86 1.92

0.40 2.13 0.60 2.25

MIR604 maize


0.01 0.01 0.1 0.1

1.83 1.80 1.93 1.83

0.45 1.60 0.40 1.50

trait

260/280 260/230

Table 4. Summary of Average Threshold Cycle (Ct) Values Obtaineda average Ct values, target gene DNA extraction trait OXY235 canola

amount (g) 0.1 0.2 1.0 2.0

method Fast ID Fast ID CTAB CTAB

HCN92 canola

0.1 0.2 1.0 2.0

DP305423 soybean

0.01% 37.85 37.73 37.21 36.94

CGC ± 0.64 ± 1.09 ± 1.00 ± 0.99


40.20 36.33 38.69 38.09

± ± ± ±

0.1 0.2 1.0 2.0


38.00 36.97 35.87 36.05

A2704-12 soybean

0.1 0.2 1.0 2.0


MIR604 maizeb

0.1 0.2 1.0 2.0


0.1% 38.92 37.80 38.85 38.36

GIPSA ± 0.76 ± 1.54 ± 0.59 ± 1.70

CGC 33.23 ± 0.90 33.22 ± 0.30 32.40 ± 0.37 32.82 ± 0.35

1.41 1.39 0.67 0.45

38.32 38.05 38.94 38.52

± ± ± ±

1.27 1.17 0.84 0.92

33.97 34.58 34.23 34.68

± ± ± ±

0.40 0.36 0.47 0.36

34.22 33.90 35.69 36.12

± ± ± ±

0.49 0.17 0.48 0.34

± ± ± ±

0.83 0.90 0.57 0.40

36.95 36.08 37.22 36.95

± ± ± ±

0.87 0.19 0.57 0.31

33.31 33.40 32.36 33.11

± ± ± ±

0.25 0.20 0.15 0.27

33.13 32.84 33.95 33.66

± ± ± ±

0.67 0.18 0.24 0.17

36.10 34.15 35.21 34.90

± ± ± ±

0.39 0.73 0.52 0.79

33.95 34.43 34.62 34.28

± ± ± ±

0.41 0.50 0.51 0.30

32.41 31.42 31.04 31.35

± ± ± ±

0.26 0.33 0.40 0.60

30.41 30.20 30.53 31.36

± ± ± ±

0.24 0.07 0.30 0.21

38.70 38.69 37.93 38.54

± ± ± ±

0.95 (83%) 0.77 (67%) 0.51 0.65 (83%)

38.51 39.82 38.57 38.91

± ± ± ±

0.20 1.35 0.67 0.69

36.20 35.91 34.93 35.83

± ± ± ±

0.72 0.91 0.19 0.42

36.14 35.77 35.79 35.43

± ± ± ±

0.36 0.23 0.57 0.40

(50%) (67%) (67%) (67%)

GIPSA 33.37 ± 0.48 32.89 ± 0.16 34.15 ± 0.11 34.02 ± 0.14

The ± standard deviation values are for 6 PCR results (2 DNA extractions × 3 PCRs). Fast ID, Fast ID DNA extraction; CTAB, CTAB DNA extraction. bThe values in parentheses indicate the percentage detected of six samples (e.g., 83% indicates that five of the six PCR samples produced detectable PCR signal and one sample did not produce detectable signal; the Ct values are the averages of detectable signals).

a

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Figure 1. Distribution of Ct values from detection of two GM events in canola (HCN92 and OXY235) fortified at two levels (0.1 and 0.01% w/w), based on two DNA extraction methods (Fast ID and CTAB) conducted at two laboratories (CGC and USDA/GIPSA).

observed in DNA extractions using the Fast ID method on samples of canola, flax, and soybean even though this did not appear to affect PCR amplification.11 Purification of 20 μg of CTAB DNA using a DNA Clean & Concentrator 25 kit (Zymo Research, Cedarlane Laboratories Ltd., Bulington, ON, Canada) from four canola, four soybean, and two maize samples was performed to determine the percentage of recovery at the CGC. Average DNA recoveries of 47, 74, and 80% were obtained for soybean, canola, and maize, respectively. The purification step

Both laboratories used a constant mass of 100 ng of DNA quantified with a PicoGreen assay for the PCR. The Abs260/280 and Abs260/230 ratios of different DNA extractions were determined at the CGC. Most of the CTAB and Fast ID extracted DNA had relatively high Abs260/280 ratios indicative of suitable DNA quality for PCR (Table 3). The DNA extracted with the Fast ID method had low Abs260/ 230 values indicating the possible presence of contaminants that absorb at 230 nm. Low Abs260/230 ratios have been 4353



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Figure 2. Distribution of Ct values from detection of two GM events in soybean (A2704 and DP305) fortified at two levels (0.1 and 0.01% w/w), based on two DNA extraction methods (Fast ID and CTAB) conducted at two laboratories (CGC and USDA/GIPSA).

generated by PCR for the 0.01% fortified samples (Figures 1−3). In general, detection of a low-level presence of GE events, such as 0.01% (1 in 10000), is relatively more challenging as the error rate due to DNA dilution, pipeting, etc., is amplified.12 For the 0.01% fortified samples and Fast ID extraction method, 0.2 g extraction samples had relatively lower Ct values (indicating a greater chance of detection) than 0.1 g extraction samples for HCN92 canola, DP305423 soybean, and A2704 soybean GE events (CGC; Table 4; Figures 1 and 2).

used for CTAB-extracted DNA may have also contributed to the variation in DNA yield. Small-Scale versus Large-Scale DNA Extractions for the Detection of LLP of GE Events. Comparable PCR results (in terms of Ct values) were obtained from DNA extracts using small-scale (Fast ID) and large-scale (CTAB) extraction methods. Higher Ct values were obtained for 0.01% fortified samples compared with 0.1% fortified samples as expected (Table 4). However, there was more variability among the Ct values 4354



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Figure 3. Distribution of Ct values from detection of GM event (MIR604) in maize fortified at two levels (0.1 and 0.01% w/w), based on two DNA extraction methods (Fast ID and CTAB) conducted at two laboratories (CGC and USDA/GIPSA).

This may potentially explain why we could not reliably detect 0.01% MIR604 maize samples. In a collaborative trial validation study, 100% detection of 0.02% LLRice62 and MS8 canola (45 samples for each event were correctly identified) was reported.14 For homogeneously prepared samples, GE trait testing laboratories can detect low concentration levels of GE events. Genetically engineered grains/ seeds are not homogeneously distributed in a grain lot.6,18 Thus, sampling is more of a challenge compared to detection of the GE traits using PCR. Results of Statistical Analysis. With respect to statistical difference in Ct values between extraction sample amounts within Fast ID (0.1 vs 0.2 g) and CTAB (1 vs 2 g), OXY235 canola samples showed no difference for both methods, at both fortification levels and across both the CGC and USDA/GIPSA laboratories; the only exception was the 0.1% Fast ID method for the USDA/GIPSA laboratory (Table 5). For HCN92 canola, CGC results showed statistical differences for Fast ID extractions at both 0.1 and 0.01% fortifications, but no differences were seen in USDA/GIPSA results. With MIR604 maize, CGC results showed differences for CTAB extractions at the 0.1% fortification. Similar results were seen for A2704 soybean and DP305423 soybean, where one laboratory may see statistical difference but results from the other laboratory showed no difference. Although the majority of comparisons across the 0.1 and 0.01% fortification levels in both laboratories showed no statistical difference within Fast ID (0.1 and 0.2 g) and CTAB (1 and 2 g) DNA extraction methods for observed Ct values, variability for reproducibility of respective Ct values may be significant for some traits with respect to combinations of fortification level, DNA extraction method, and amount of extraction sample.

On the basis of these data, improvement in the detection of the low-level presence of GE events may not necessarily be achieved as a result of using large-scale DNA extraction methods such as CTAB. The use of small-scale DNA extraction methods saves time and money. For the small-scale extraction method, sufficient amounts of DNA were achieved for replicate PCR and repeatable Ct values were obtained, especially for 0.1% fortified samples. Holden et al. suggested that samples prepared at the 0.1% fortification level can generate false-negative results when certain detergent-based DNA isolation methods are used.13 The bulk of the false-negative results were attributed to the presence of inhibitors that inactivate Taq DNA polymerase in the PCR. For the MIR604 maize event, repeatable and consistent detection by PCR was not achieved at the 0.01% fortification level regardless of the extraction method or the amount of starting material used under the conditions of this study design. In addition, significant variability was observed, including falsenegative results, between the two DNA extraction methods as well as within replicates for the Ct values obtained (Table 4). The MIR604 heterozygous reference maize purchased from AOCS was reported to have a purity of 99.98%; thus, this may have also affected the fortification and in turn the detection of the MIR604 maize event at 0.01% level. Haploid genome weight for Brassica napus (canola), maize, and soybean has been reported to be 1.13 pg,15 2.3 pg,16 and 1.12 pg,17 respectively. On the basis of the described genome weights, 100 ng of DNA spiked at the 0.01% level will have approximately nine copies of OXY235 canola and DP305423 soybean and four copies of maize. Thus, the chance of detection of GE canola and soybean samples at low levels is higher than that of GE maize samples. 4355



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Table 5. Probability of Statistical Difference in Observed Ct Values within Fast ID (0.1 vs 0.2 g Samples) and CTAB (1 vs 2 g Samples) DNA Extraction Methods at 0.1 and 0.01% Fortification Levels Conducted at Two Laboratories (Kruskall−Wallis Test) CGC laboratory trait OXY235 canola

HCN92 canola

4.3485 2.3222 2.0769 0.2308

0.0370 0.1275 0.1495 0.6310

5.0256 2.5641 7.5000 2.0842

0.0250 0.1093 0.0062 0.1488

0.8333 1.6468 0.0256 0.6410

0.3613 0.1994 0.8728 0.4233


8.3077 1.8591 8.3077 0.6410

0.0039 0.1727 0.0039 0.4233

3.1466 5.3330 2.0842 0.7500

0.0761 0.0209 0.1488 0.3865


0.6410 8.3077 3.1026 0.4103

0.4233 0.0039 0.0782 0.5218

0.0581 4.3485 3.6923 0.6433

0.8095 0.0370 0.0547 0.4225


1.2564 8.3368 0.0600 2.7000

0.2623 0.0039 0.8065 0.1003

3.6923 0.9231 0.4286 0.0000

0.0547 0.3367 0.5127 1.0000

Pr > chi-square

0.10


0.0000 3.4029 0.1333 0.1026


0.10

0.10

0.10 0.01

MIR604 maize

1.0000 0.0651 0.7150 0.7488

chi-square

0.01

DP305423 soybean

Pr > chi-squarea

DNA extraction method

0.01

A2704 soybean

chi-square


0.01

0.10 0.01

GIPSA laboratory a

Difference within DNA extraction methods for each % GM fortification levels are deemed statistically significant where Pr > chi-square is ≤0.05 for 95% confidence. a

Table 6. Probability of Statistical Difference between Fast ID and CTAB DNA Extraction Methods for Observed Ct Values from Real-Time PCR for 0.1 and 0.01% Fortification Levels with Five Traits Conducted at Two Labs (Kruskall−Wallis Test) CGC laboratory trait


GIPSA laboratory

chi-square

Pr > chi-squarea

chi-square

Pr > chi-squarea

OXY235 canola

0.10 0.01

6.166 3.8788

0.0130 0.0489

13.4584 0.2409

0.0002 0.6235

HCN92 canola

0.10 0.01

0.7507 0.1857

0.3863 0.6665

16.5082 1.1413

chi-square is ≤0.05 for 95% confidence. a

0.1% fortification, whereas for HCN92 canola only USDA/ GIPSA results showed statistical differences at the 0.1% fortification level. Both laboratories found statistical differences between extraction methods for A2704 soybean fortified at the 0.1% level. Comparisons for OXY235 canola showed statistical differences for both fortification levels in CGC results, whereas USDA/GIPSA results showed differences only for the 0.1% fortification level. For these comparisons, however, Ct results

In terms of comparing DNA extraction methods (Fast ID vs CTAB) for statistical difference in Ct values across fortification levels, differences in results between laboratories were found (Table 6). Only DP305423 soybean showed statistical difference between Fast ID and CTAB for both 0.1 and 0.01% fortification levels across both CGC and USDA/ GIPSA laboratories. For MIR604 maize only CGC results showed statistical differences between extraction methods at 4356



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were aggregated across sample extraction amounts, and variance in Ct values due to different amounts of starting material used within each extraction method likely contributed to the observed variance between laboratories. Summary. In this study, the influence of starting material on the detection of low-level presence of GE traits was investigated using five GE events (two events each for canola and soybean and one event for maize). Higher DNA yields were obtained with the larger scale DNA extraction method (1.0 and 2.0 g samples) compared to the smaller scale DNA extraction method (0.1 and 0.2 g samples). There was variability in the DNA yield obtained between the CGC and USDA/GIPSA laboratories involved in this study, especially for the large-scale DNA extraction method. CTAB-extracted DNA that was further purified on a DNA Clean & Concentrator Kit had greater purity (based on Abs260/280 and Abs260/ 230 ratios) compared with the Fast ID extracted DNA. When the large-scale CTAB DNA extraction method (1.0 and 2.0 g samples) was compared with a small-scale Fast ID DNA extraction method (0.1 and 0.2 g samples), the data did not reveal differences in terms of detection by PCR for four of the five GE events studied at 0.1 and 0.01% fortification levels. Overall, reliable detection of the GE events was achieved at the 0.1% level. Reliable detection at the 0.01% fortification level was achieved for the four canola and soybean GE traits, but not as reliably for the MIR604 maize GE trait. The 0.01% 100 ng maize DNA has a low MIR604 copy number, which may have resulted in unreliable detection. There was more variability in Ct values generated by the duplicate DNA extractions for the 0.01% fortified samples using 0.1 g of starting material. Thus, detection of samples at this fortification level using only 0.1 g of starting material could potentially produce false-negative results in maize but not in soybean or canola. In general, a kit-based DNA extraction method (using 0.1 and 0.2 g of sample) can be used instead of the large-scale CTAB DNA extraction method for the detection of low levels of GE events in ground grain samples, which could save time and cost.

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REFERENCES

(1) Demeke, T.; Perry, D. J.; Scowcroft, W. R. Adventitious presence of GMOs: scientific overview for Canadian grains. Can. J. Plant Sci. 2006, 86, 1−23. (2) Grohmann, L.; Busch, U.; Pecoraro, S.; Hess, N.; Pietsch, K.; Mankertz, J. Collaborative trail validation of a construct-specific realtime PCR method for detection of genetically modified linseed event ‘CDC Triffid’ FP967. Eur. Food Res. Technol. 2011, 232, 557−561. (3) Davison, J. GM plants: science, politics and EC regulations. Plant Sci. 2010, 178, 94−98. (4) EU Commission Regulation No. 619/2011. Laying down the methods of sampling and analysis for the official control of feed as regards presence of genetically modified material for which an authorization procedure is pending or the authorization of which has expired. Off. J. Eur. Union 2011 (June 24), L166/9−L1669/15. (5) Nakamura, K.; Akiyama, H.; Yamada, C.; Satoh, R.; Nakiyama, D.; Sakata, K.; Kawakami, H.; Mano, J.; Kitta, K.; Teshima, R. Novel method to detect a construct-specific sequence of the acetolactate synthase gene in genetically-modified flax CDC Triffid (FP967). Biol. Pharm. Bull. 2010, 33, 532−534. (6) Lipp, M.; Shillito, R.; Giroux, R.; Spiegelhalter, F.; Charlton, S.; Pinero, D.; Song, P. Polymerase chain reaction technology as analytical tool in agricultural biotechnology. J. Agric. Food Chem. 2005, 88, 136− 155. (7) Waiblinger, H.-U.; Graf, N.; Broll, H.; Grohmann, L.; Pietsch, K. Evaluation of real-time PCR results at the limit of detection. J. Verbr. Lebensm. 2011, 6, 411−417. (8) Demeke, T.; Ratnayaka, I.; Phan, A. Effects of DNA extraction and purification methods on real-time quantitative PCR analysis of Roundup Ready soybean. J. AOAC Int. 2009, 92, 1136−1144. (9) Juan, J. S.; Reed, A.; Chen, F.; Stewart, C. C., Jr. Statistical analysis of real-time PCR data. BMC Bioinformatics 2006, 7, 85; http://www.biomedcentral.com/1471-2105/7/85 (accessed January 24, 2014). (10) SAS Institute. SAS/STAT(R) 9.2 User’s Guide, 2nd ed., release 9.2; SAS Institute Inc.: Cary, NC, USA, 2013; http://support.sas.com/ documentation/cdl/en/statug/63033/HTML/default/viewer. htm#statug_npar1way_sect004.htm (accessed January 24, 2014). (11) Demeke, T.; Ratnayaka, I.; Holigroski, M.; Phan, A. Assessment of DNA extraction methods for PCR testing of discontinued or unapproved biotech events in single seeds of canola, flax and soybean. Food Control 2012, 24, 44−49. (12) Booker, H. M.; Lamb, E. G. Quantification of low-level GM seed presence in Canadian commercial flax stocks. AgBioForum 2012, 15, 31−35. (13) Holden, M. J.; Blasic, J. R.; Bussjaeger, L.; Kao, C.; Shokere, L. A.; Kendall, D. C.; Freese, L.; Jenkins, G. R. Evaluation of extraction methodologies for corn kernel (Zea mays) DNA for detection of trace amounts of biotechnology-derived DNA. J. Agric. Food Chem. 2003, 51, 2468−2474. (14) Grohmann, L.; Brünen-Nieweler, C.; Nemeth, A.; Waiblinger, H.-U. Collaborative trial validation studies of real-time PCR-based GMO screening methods for detection of the bar Gene and the ctp2cp4epsps construct. J. Agric. Food Chem. 2009, 57, 8913−8920. (15) Johnston, J. S.; Pepper, A. E.; Hall, A. E.; Chen, Z. J.; Hodnett, G.; Drabek, J.; Lopez, R.; Price, H. J. Evolution of genome size in Brassicaceae. Ann. Bot. 2005, 95, 229−235. (16) Schnable, P. S.; Ware, D.; Fulton, R. S.; Stein, J. C.; Wei, F.; Pasternak, S.; Liang, C.; Zhang, J.; Fulton, L.; Graves, T. A.; et al. The B73 maize genome: complexity, diversity, and dynamics. Science 2009, 326, 1112−1115. (17) Arumuganthan, K.; Earle, E. D. Nuclear DNA content of some important plant species. Plant Mol. Biol. Rep. 1991, 93, 208−218. (18) Paoletti, C.; Heissenberger, A.; Mazarra, M.; Larcher, S.; Grazioli, E.; Corbisier, P.; Hess, N.; Berben, G.; Lübeck, P. S.; De Loose, M.; Moran, G.; Henry, C.; Brera, C.; Folch, I.; Ovesna, J.; Van den Eede, G. Kernel lot distribution assessment (KeLDA): a study on the distribution of GMO in large soybean shipments. Eur. Food Res. Technol. 2006, 224, 129−139.

AUTHOR INFORMATION

Corresponding Author

*(T.D.) Phone: (204) 984-4582. Fax: (204) 983-0724. E-mail: [email protected]. Notes ∥

Paper No. 1076 of the Grain Research Laboratory, Canadian Grain Commission. The authors declare no competing financial interest.

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ACKNOWLEDGMENTS Paul Wacker is acknowledged for help in carrying out the experiments at USDA/GIPSA. Stefan Wagener (Canadian Grain Commission, Grain Research Laboratory) is acknowledged for reviewing the manuscript.

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ABBREVIATIONS USED Ct, cycle threshold value; CGC, Canadian Grain Commission; CTAB, cetylmethyl-ammonium bromide; GE, genetically engineered; SDS, sodium dodecyl sulfate; USDA/GIPSA, U.S. Department of Agriculture, Grain Inspection, Packers and Stockyards Administration 4357



Article

(19) EURL GMFF (European Union Reference Laboratory for Genetically Modified Food and Feed (2011). Event-specific method for the quantification of oilseed rape Topas 19/2using real-time PCR; http://gmo-crl.jrc.ec.europa.eu/summaries/CRLVL1204%20VP.pdf (accessed January 24, 2014). (20) Wu, G.; Wu, Y.; Xiao, L.; Lu, C. Event-specific qualitative and quantitative PCR methods for the detection of genetically modified rapeseed Oxy-235. Transgenic Res. 2008, 17, 851−862. (21) EURL GMFF (European Union Reference Laboratory for Genetically Modified Food and Feed (2009). Event-specific method for the quantification of soybean event DP-305423-1 using real-time PCR; http://gmo-crl.jrc.ec.europa.eu/summaries/ CRLVL0707VP%20Corr2%20EURL%20web.pdf (accessed January 24, 2014). (22) EURL GMFF (European Union Reference Laboratory for Genetically Modified Food and Feed (2009). Event-specific method for the quantification of soybean line A2704−12 using real-time PCR; http://gmo-crl.jrc.ec.europa.eu/summaries/A2704-12_soybean_ validated_Method.pdf (accessed January 24, 2014). (23) EURL GMFF (European Union Reference Laboratory for Genetically Modified Food and Feed (2009). Event-specific method for the quantification of maize line MIR604 using real-time PCR; http://gmo-crl.jrc.ec.europa.eu/summaries/MIR604_validated_ Method_correctedversion1.pdf (accessed January 24, 2014).

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