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1-6-29 Minami-machi, Minami-ku, Hiroshima 734-0007, Japan. 2National Institute of ... detection system was developed for Roundup Ready®. Soybean (RRS) ...
Biosci. Biotechnol. Biochem., 70 (4), 821–827, 2006

Quantification of Genetically Modified Soybeans Using a Combination of a Capillary-Type Real-Time PCR System and a Plasmid Reference Standard Akie T OYOTA,1;4 Hiroshi A KIYAMA,2; y Mitsunori S UGIMURA,1 Takahiro WATANABE,2 Hiroyuki K IKUCHI,2 Hisayuki K ANAMORI,1 Akihiro H INO,3 Muneharu E SAKA,4 and Tamio M AITANI2 1

Hiroshima Prefectural Institute of Public Health and Environment, 1-6-29 Minami-machi, Minami-ku, Hiroshima 734-0007, Japan 2 National Institute of Health Sciences, 1-18-1 Kamiyoga, Setagaya-ku, Tokyo 158-8501, Japan 3 National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan 4 Graduate School of Biosphere Sciences, Hiroshima University, 1-4-4 Kagamiyama, Higashi-Hiroshima, Hiroshima 739-8528, Japan Received August 23, 2005; Accepted December 15, 2005

Because the labeling of grains and feed- and foodstuffs is mandatory if the genetically modified organism (GMO) content exceeds a certain level of approved genetically modified varieties in many countries, there is a need for a rapid and useful method of GMO quantification in food samples. In this study, a rapid detection system was developed for Roundup Ready Soybean (RRS) quantification using a combination of a capillary-type real-time PCR system, a LightCycler real-time PCR system, and plasmid DNA as the reference standard. In addition, we showed for the first time that the plasmid and genomic DNA should be similar in the established detection system because the PCR efficiencies of using plasmid DNA and using genomic DNA were not significantly different. The conversion factor (Cf ) to calculate RRS content (%) was further determined from the average value analyzed in three laboratories. The accuracy and reproducibility of this system for RRS quantification at a level of 5.0% were within a range from 4.46 to 5.07% for RRS content and within a range from 2.0% to 7.0% for the relative standard deviation (RSD) value, respectively. This system rapidly monitored the labeling system and had allowable levels of accuracy and precision. Key words:

genetically modified soybean; Roundup Ready Soybean; capillary-type real-time PCR system

The production of genetically modified (GM) crops, especially Roundup Ready Soybean (RRS), has increased in the United States over the past several years.1) In some countries, controversial issues still exist y

regarding the acceptance of GM crops, and concerns about their safety persist in public opinion. In many countries, the labeling of grains and feed- and foodstuffs is mandatory if the genetically modified organism (GMO) content exceeds a certain level of approved GM varieties. For instance, the European Union (EU), Japan, Korea, and Taiwan have set threshold values at 0.9% (EU Regulation No. 1830/2003), 5%, 3%, and 5% respectively of GMO material in a non-GM background as the basis for labeling.2–4) The enforcement of these threshold values has created a demand for the development of reliable GMO analysis methods of a rapid and inexpensive character. Most of the established analytical methods for detecting the GMO identification and quantification in foods are based on the polymerase chain reaction (PCR), due to its sensitivity, specificity, and applicability to the analysis of complex food matrices.5–18) Furthermore, many real-time PCR systems based on fluorescent detection, such as TaqMan chemistry, have been developed to identify and quantify GM soybeans, GM maize, and GM varieties of other agricultural commodities.19–28) Real-time PCR systems using TaqMan chemistry are based on the use of a fluorescent TaqMan probe that monitors the formation of the PCR product during each cycle of the reaction. In addition, most commonly, GMO quantification by quantitative real-time PCR methods is calculated from the ratio of the target transgenic specific DNA sequence copy number vs. the DNA sequence copy number of the respective target plant species (taxon gene sequence). Determination of the copy number by real-time PCR methods involves the establishment of calibration curves based on analysis of

To whom correspondence should be addressed. Tel: +81-3-3700-9397; Fax: +81-3-3707-6950; E-mail: [email protected]

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a set of calibrators such as genomic DNA (gDNA) or plasmid DNA (pDNA). Especially, plasmid DNA markers containing a cloned transgenic sequence have been used and are increasingly promoted as the standards of choice for GMO analysis, because pDNA is semi-infinitely available at the same quality. We have already developed quantification methods based on the TaqMan Chemistry of real-time PCR for five lines of GM maize and a GM soybean, Roundup Ready , using pDNA as the reference molecules, and our methods have been validated by interlaboratory testing.23,24) These methods were performed using an ABI PRISM 7700 real-time PCR system, titer-plate type equipment. However, if these methods were to be applied to other real-time PCR equipment, beforehand we had to set up the PCR conditions and determine the conversion factor (Cf ), and the coefficient value to calculate the GMO amount (%). Because titer-plate type PCR equipment is time-consuming in PCR and comparatively expensive, the application of rapid and inexpensive equipment is desirable. The LightCycler real-time PCR system, capillary-type real-time PCR equipment, is one of the most widely used types of equipment in biomaterial tests and is more rapid due to the temperature change using air media, and is also comparatively inexpensive.29,30) In the present study, we developed a detection system for RRS quantification using a combination of reference plasmid DNA, a LightCycler real-time PCR system, and capillary-type real-time PCR equipment, and evaluated the method developed, including determination of the Cf , to calculate the RRS content (%) system. In addition, we showed the equivalence of pDNA and gDNA as reference standards in the method developed using the LightCycler real-time PCR system.

Materials and Methods Materials. Non-GM soybean grains were obtained from the Ministry of Health, Labour, and Welfare (MHLW) of Japan. RRS seeds were kindly provided by Monsanto (St. Louis, MO). Soybean powder certified reference material (CRM) IRMM-5, 5% RRS, was purchased commercially (Fluka, Buchs SG, Switzerland). Three soybean grain samples labeled as non-GM were purchased commercially in Tokyo. As a standard material for the calibration curve, RRS Detection Plasmid Set-ColE1/TE (Nippon Gene, Toyama, Japan) was used. Preparation of test samples. To prepare GM mixed test samples, soybeans (GM seeds and non-GM seeds) were separately milled to a fine powder using grinders (Retsch, Haan, Germany), and then mixed with 1% and 5% GM soybeans weight per weight, respectively, according to a previously reported procedure.23) Extraction and purification of gDNA. DNA extraction

and purification was carried out using an anion exchange resin-type kit (Genomic-tip 20/G; Qiagen, Hilden, Germany) according to the manufacturer’s manual, with the following modification: Powdered samples (500 mg) were vortexed in 15 ml of Digestion Buffer G2 (800 mM guanidine HCl; 30 mM Tris–Cl, pH 8.0; 30 mM EDTA, pH 8.0; 5% Tween-20; 0.5% Triton X-100) and 200 ml of (1 mg/ml) -amylase, and then incubated at 37  C for 1 h. One hundred ml of (20 mg/ml) Proteinase K and 20 ml of (100 mg/ml) RNase A were added to the samples and then incubated at 50  C for 2 h. The supernatant generated after centrifugation at 8;000  g for 25 min at 4  C was filtered through Millex-HV (Millipore, Billerica, MA). Two ml of the supernatant was applied three times to a Genomic-tip equilibrated with 1 ml of Buffer QBT (750 mM NaCl; 50 mM MOPS, pH 7.0; 15% isopropanol; 0.15% Triton X-100). The tip was washed three times with 2 ml of Buffer QC (1.0 M NaCl; 50 mM MOPS, pH 7.0; 15% isopropanol), and then eluted twice with 1 ml of Buffer QF (1.25 M NaCl; 50 mM Tris–Cl, pH 8.5; 15% isopropanol; at 50  C). The eluate was mixed with 0.1 volume of 3 M sodium acetate at pH 4.8 and 2.5 volumes of ethanol. The mixture was centrifuged at 8;000  g for 20 min at 4  C. The supernatant was discarded and the pellets washed with 70% ethanol. Finally, the DNA was dissolved in 200 ml of TE buffer (10 mM Tris–Cl, pH 8.0; 1 mM EDTA). The DNA concentration in the solutions was determined by measuring UV absorption at 260 nm using a GeneQuant pro spectrophotometer (Amersham Biosciences, Piscataway, NJ). The purity of the extracted DNA was evaluated using a ratio of 260/280 nm, and the ratio was between 1.7 and 2.0 for most of the test samples. The extracted DNA was diluted with an appropriate volume of DW to a final concentration of 10 ng/ml and stored at 20  C until used. These DNA samples were used for the subsequent PCR analysis. Real-time quantitative PCR of the endogenous lectin gene (Le1) and the RRS specific gene (RRS). Fluorescence resonance energy transfer (FRET) hybridization probes were used for quantification in the LightCycler real-time PCR system (Roche Diagnostics, Mannheim, Germany). The lectin gene (Le1) was chosen as the reference DNA gene for quantitative analysis, because Le1 has been reported as a single copy gene in soybean and is widely used in the previously reported methods. The GM Soybean (RRS) Detection Le1 Oligonucleotide Set and GM Soybean (RRS) Detection RRS Oligonucleotide Set (Nippon Gene) were used for Le1 and RRS specific gene (RRS) assays as the primers and probes. PCR was performed in glass capillary tubes (Roche). Unless otherwise specified, the total reaction volume of 20 ml contained 50 ng of the DNA template, 0.25 mM of each primer, 0.2 mM of the probe, 4.0 mM of MgCl2 , and a LightCycler-FastStart DNA Master Hybridization Probes Kit (Roche), which contained FastStart Taq DNA polymerase, hybridization probes for detection by

Quantification of Genetically Modified Soybeans 

the LightCycler real-time PCR system, reaction buffer, and dNTPs. The cycling conditions were as follows: preincubation at 95  C for 10 min, denaturation at 95  C for 15 s (20  C/s), annealing at 59  C for 30 s (1  C/s), and pre-incubation at 95  C for 10 min, denaturation at 95  C for 15 s (1  C/s), and annealing at 59  C for 30 s (20  C/s). Both cycles were repeated 50 times. Standard curves were calibrated using five concentrations of control plasmids 40, 250, 3,000, 40,000, and 500,000 copies per reaction. A no-template control was also prepared as the negative control. Each control sample was run in one capillary for each target, and unless otherwise specified, each soybean sample was run in duplicate for each target. The data were analyzed using LightCycler real-time PCR system Data Analysis software version 3.5.5 and the ‘‘Fit Points’’ algorithm. The cycle at which the amplification curve crosses the threshold was defined as Ct (cycle of threshold), and the standard curve was constructed from the mean Ct values of the triplicate determination. Measurement of PCR efficiency. The PCR efficiency was calculated using the slope of the standard curve according to Formula1 as follows.28) Formula1: PCR efficiency ¼ 10ð1=slopeÞ Measurement of conversion factor and calculation of GMO amount. The copy number of each sample was obtained as the mean value of triplicates compared to the optimal standard curve.23,24) The ratio of the copy number of RRS and Le1 in each genuine seed was calculated using Formula2 below and was defined as the conversion factor (Cf ). The GMO amounts (%) were calculated using Formula3 below and the defined Cf . Formula2: Cf ¼ ðcopies of RRS in the DNA extracted from GM seedsÞ=ðcopies of Le1 in the DNA extracted from GM seedsÞ Formula3: GMO amount (%) ¼ ðcopies of RRS in the DNA extracted from an unknown sample  100Þ=ðcopies of Le1 in the DNA extracted from an unknown sample  Cf Þ Statistical analysis. All values are expressed as means  standard deviation of the mean. Statistical comparisons were performed by Student’s t-test. In all cases, probability (P) values below 0.05 were considered significant.

Results and Discussion Optimization of PCR conditions To optimize real-time PCR conditions using the LightCycler real-time PCR system, specific primers,

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probes, and pDNA, we examined several factors such as MgCl2 concentration and the speed of the temperature change between the annealing temperature and the denaturing temperature under the PCR conditions. Le1 and RRS of the gDNA extracted from soybean grain flour were determined using the control pDNA as the reference standard. The optimal MgCl2 concentrations in the PCR reaction were investigated at a concentration of 2.0 mM to 4.0 mM. With increasing MgCl2 concentration in the PCR reaction, the Ct of both Le1 and RRS gradually decreased and the detection range appeared to be expanded. Hence, we concluded that the optimal MgCl2 concentration is 4.0 mM in each reaction buffer. To optimize the amplification curves, we examined the speed of the temperature change between the denaturing temperature and the annealing temperature under the PCR conditions using the LightCycler realtime PCR system. As shown in Fig. 1, among the several conditions examined, satisfactory amplification curves were observed when the speed of the rising step from the annealing temperature to the denaturing temperature was slightly rapid (1  C/s) and the speed of the cooling step from the denaturing temperature to the annealing temperature was even faster (20  C/s). Analysis of PCR efficiencies for gDNA and pDNA using the established real-time quantitative system We have reported the real-time PCR detection method for construct-specific quantification of GM soybeans and maize using the reference pDNA as a standard molecule and an ABI PRISM 7700 system as the real-time PCR equipment,23) but comparable studies between pDNA and gDNA have not yet been performed. We concluded that these studies are important, because comparable behavior between pDNA and gDNA in the PCR is reflected in the characteristics of the standard curves obtained. Hence, to assess the validity of the reference pDNA for quantitative analysis of GM soybeans, we compared the PCR efficiencies of the pDNA and gDNA (Formula1). The standard curves of pDNA were calibrated by the pDNA series (five concentrations of the control plasmids 40, 250, 3,000, 40,000, and 500,000 copies per reaction). The standard curves of gDNA were calibrated by the gDNA series (five concentrations of diluted DNA extracted from 100% RRS soy 0.005, 0.05, 0.5, 5, and 50 ng per reaction). As shown in Table 1 and Fig. 2, the average calculated R2 values of the standard curves using pDNA for Le1 and RRS were 0.9993 and 0.9999 respectively. The average calculated R2 values of the standard curves using gDNA for Le1 and RRS were 0.9980 and 0.9999 respectively. Since approximately 50,000 copies of Le1 are contained in 50 ng of soybean DNA, 40 copies of RRS in 50 ng of soybean DNA correspond to a GMO content of approximately 0.1%. These results suggest that the detection limit is 0.1%. As shown in Fig. 2, the pDNA and gDNA standard curves show nearly equal slopes for Le1 and RRS. Table 2

A. TOYOTA et al.

Fluorescence F1/F2

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10

B

A

a b c d e

f g h

0.1 0.06 10

50

i

j

10

50

Fluorescence F1/F2

Cycle number 10

C

D

a b

c

f g h i j

d e

0.1 0.06

50

10

10

50

Cycle number Fig. 1. Amplification Plots of PCR Product from pDNA and gDNA Using the LightCycler Real-Time System. Le1 amplification plots from pDNA (A) and gDNA (B); RRS amplification plots from pDNA (C) and gDNA (D). a, 500 k copies; b, 40 k copies; c, 3 k copies; d, 250 copies; e, 40 copies; f, 50 ng; g, 5 ng; h, 0.5 ng; i, 0.05 ng and j, 0.005 ng.

Table 1. Quantitative Results for the Control Plasmid and the RRS Genome

a

Sample

Target

Standard (copies)

Cta

SDb

Sample

Target

Standard (ng)

Cta

SDb

pDNA

Le1

40 250 3,000 40,000 500,000

35.02 32.66 28.79 24.83 21.34

0.63 0.82 0.65 0.77 0.85

gDNA

Le1

0.005 0.05 0.5 5 50

37.58 34.83 30.99 27.59 24.20

1.16 1.00 0.38 0.38 0.35

pDNA

RRS

40 250 3,000 40,000 500,000

32.93 30.39 26.58 22.82 19.17

0.83 0.74 0.58 0.61 0.69

gDNA

RRS

0.005 0.05 0.5 5 50

35.94 32.64 29.31 25.90 22.43

1.10 0.65 0.38 0.33 0.21

Ct, cycle of threshold (mean of six replicates for standard curves). b SD, standard deviation.

shows a statistical comparative analysis of the calibration curves set up with pDNA and gDNA using the established detection system. As shown in this table, it is clear that the slopes of gDNA and pDNA are not significantly different for either Le1 or RRS. The average PCR efficiencies for pDNA and gDNA were 1.964 and 1.953 in Le1 and 1.980 and 1.996 in RRS respectively. These results suggest that pDNA and gDNA behave in a similar way in the established detection system. Determination of Cf To calculate the RRS content (%) according to Formula2, described in ‘‘Materials and Methods,’’ we had to determine the conversion factor (Cf ) as described in Formula2.23) The measurements of Cf in DNA extracted from 100% RRS were performed by different researchers from three laboratories using the LightCycler real-time PCR system equipment set in each laboratory, and were run in triplicate for Le1 and RRS. The Cf values determined in the three laboratories were

0.78, 0.88, and 0.84. The average value obtained from the three laboratories was defined as the Cf for calculation of RRS content (%) in the soybean samples. The averaged Cf for the RRS quantification was 0.83. The relative standard deviation (RSD) value of the Cf was 5.5%. Accuracy, repeatability, and reproducibility for the quantification of RRS in soybean samples The accuracy, repeatability, and reproducibility of RRS quantification using the established detection method were assessed. The DNA extracted from a 5% RRS mixed sample was repeatedly amplified five times a day. Each sample was run in duplicate for Le1 and RRS. The RRS content (%) of each sample was calculated using Formula3, described in ‘‘Materials and Methods.’’23) Table 3 shows the results of this reproducibility study of the calculated RRS content (%) for the same day and three different days using the established method. As can be seen, the calculated RRS

Quantification of Genetically Modified Soybeans

A

B

Le1 (pDNA)

40

Ct

Ct

E=1.96

30 y = −3.3876x + 40.575 R2 = 0.9993

25

y = −3.3987x + 30.014 R2 = 0.998

20 0

2

4 Log copies

−3

6

RRS (pDNA)

D

−2

−1 0 Log ng

1

2

RRS (gDNA)

40 35

30

Ct

E=1.98

Ct

E=1.95

30 25

20

35

Le1 (gDNA)

40 35

35

C

825

25

E=2.00

30 25

y = −3.3767x + 38.384 R2 = 0.9999

20

y = −3.3763x + 28.229 R2 = 0.9999

20

15

15 0

2

Log copies

4

6

−3

−2

−1 0 Log ng

1

2

Fig. 2. Standard Curves of PCR Products Amplified from pDNA Series and RRS gDNA Series. Le1 standard curves obtained from pDNA (A) and gDNA (B), RRS standard curves obtained from pDNA (C) and gDNA (D). Six standard curves are indicated in each column. The standard curves for A and C were obtained from 40 to 500,000 copies of pDNA, and those for B and D were obtained from 0.005 to 50 ng of gDNA.

Table 2. Statistical Analysis of the Results of Calibration Curves Set up with pDNA and gDNA Using the Established Detection System Target

Criterium

Le1

slope

6 6 6 6

0:379

0.720

0.227

0.829

6 6 6 6

0.502

0.637

0:606

0.571

Variance of data set

n

pDNA gDNA pDNA gDNA

3:416 3:463 1.964 1.953

0.013 0.073 0.002 0.011

pDNA gDNA pDNA gDNA

3:377 3:344 1.980 1.996

0.019 0.035 0.003 0.005

slope E

Table 3. Accuracy, Repeatability, and Reproducibility of Quantitative Results of 5% RRS Mixing Sample Same day (n ¼ 5)

GMO Amount (%) RSD (%)

P (T < t) two-sided

Mean of data set

E RRS

T statistical data

Type of calibrator

Three days (n ¼ 15)

1

2

3

4.95 2.00

4.35 2.01

4.91 4.51

4.74 7.00

content ranges from 4.35 to 4.95% for three different days, the RSD values range from 2.01 to 4.51% for one day, and the RSD value is 7.00% for three different days. These results indicate that we detected the RRS content of samples with a good accuracy and precision by the detection system developed in this study.

Quantitative results of RRS content in the soybean samples To attempt to apply this method further to other level samples, GMO content (%) was measured using the established detection method and calculated for 0, 1.0, and 5.0% of RRS mixed samples and an IRMM-5 (5.0%) sample of a certified reference sample. As shown in Table 4, the calculated GMO contents of the 0, 1.0, and 5.0% RRS mixed samples and the IRMM-5 sample were 0, 0.86, 4.74, and 4.62% respectively. The RSD values of the 1.0% mixed sample, the 5.0% mixed sample, and the IRMM-5 sample were 22.28, 7.00, and 12.42% respectively. These results indicate that all the calculated RRS contents for these samples were very close to the expected theoretical values and are reasonable.

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Table 4. Quantitative Results of the Soybean Samples Including RRS Using the Established System

6)

GMO Amount of RRS (%) Sample

(n)

Actual

Calculated

RSD (%)

RRSO RRS1 RRS5 IRMM5

(2) (5) (15) (5)

0 1.0 5.0 5.0

0 0.86 4.74 4.62

22.28 7.00 12.42

7)

8)

Furthermore, the established method was applied to the three imported commercially available soybean samples. No RRS was detected in any of the samples, although Le1 was definitely detected (data not shown). This study shows that the previously described and established method using pDNA and ABI PRISM 7700 equipment can be adapted to a capillary-type real-time PCR system and still perform acceptably. Furthermore, we demonstrated for the first time that pDNA and gDNA can perform similarly as reference molecules. With respect to the similarity between pDNA and gDNA, we believe that these data have not been demonstrated before and that they can address the on-going criticism of amplification differences when using pDNA as a reference. In conclusion, we developed a rapid detection system for RRS quantification using a combination of the LightCycler real-time PCR system and pDNA as a reference standard. In addition, we showed that pDNA and gDNA are similar to the established PCR system and are thus commutable. This method can rapidly monitor a labeling system and has sufficient levels of accuracy and precision.

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Acknowledgment We are very grateful to Monsanto Co., USA, for providing reference RRS seed materials. We thank Dr. Doris Dixon and Mr. Hiroo Wakimori for useful suggestions, and Ms. Ai Miyazaki and Ms. Chiseko Wakui for teaching us the technique for the LightCycler real-time PCR system. This study was partly supported by a grant from the Ministry of Health, Labour, and Welfare of Japan.

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