Research Article Received: 1 October 2013
Revised: 12 February 2014
Accepted article published: 24 February 2014
Published online in Wiley Online Library:
(wileyonlinelibrary.com) DOI 10.1002/jsfa.6625
Screening DNA chip and event-specific multiplex PCR detection methods for biotech crops Seong-Hun Lee* Abstract BACKGROUND: There are about 80 biotech crop events that have been approved by safety assessment in Korea. They have been controlled by genetically modified organism (GMO) and living modified organism (LMO) labeling systems. The DNA-based detection method has been used as an efficient scientific management tool. Recently, the multiplex polymerase chain reaction (PCR) and DNA chip have been developed as simultaneous detection methods for several biotech crops’ events. RESULTS: The event-specific multiplex PCR method was developed to detect five biotech maize events: MIR604, Event 3272, LY 038, MON 88017 and DAS-59122-7. The specificity was confirmed and the sensitivity was 0.5%. The screening DNA chip was developed from four endogenous genes of soybean, maize, cotton and canola respectively along with two regulatory elements and seven genes: P35S, tNOS, pat, bar, epsps1, epsps2, pmi, cry1Ac and cry3B. The specificity was confirmed and the sensitivity was 0.5% for four crops’ 12 events: one soybean, six maize, three cotton and two canola events. CONCLUSION: The multiplex PCR and DNA chip can be available for screening, gene-specific and event-specific analysis of biotech crops as efficient detection methods by saving on workload and time. © 2014 Society of Chemical Industry Keywords: biotech crops; event-specific; multiplex PCR; screening; gene-specific; DNA chip
INTRODUCTION Since the introduction of the first genetically modified (GM) tomato, Flavr Savr™, in 1994, biotech crops have been increasingly developed and commercialized. Compared with conventional crops, biotech crops display insect resistance, herbicide tolerance or some other advantageous function. The global area for biotech crops has increased continuously from 1.7 million hectares in 1996 to 170.3 million hectares in 2012. The global adoption rates for principal biotech crops are as follows: soybean 81%, maize 35%, cotton 81% and canola 30%.1 Maize (Zea mays L.) is the most commercialized biotech crop and also the world’s third leading cereal crop after wheat and rice. It has been cultivated for human food, animal feed and biofuel. Seven events of biotech maize are typical: NK 603, MON 863, MIR 604, Event 3272, LY 038, MON 88017 and DAS-59122-7. NK 603 contains the cp4 epsps gene,2 MON 863 the cry3Bb1 gene,3 MIR 604 the mcry3A and pmi genes,4 Event 3272 the amy797E and pmi genes,5 LY 038 the cordapA gene,6 MON 88017 the cry3Bb1 and cp4 epsps genes7 and DAS-59122-7 the cry34Ab1, cry35Ab1 and pat genes.8 Cotton (Gossypium hirsutum L.) is an important economic crop used as a fiber and in cooking oil, snack foods and important protein concentrates for livestock. Three events of biotech cotton are typical: MON 531, MON 1445 and MON 15985. MON 531 contains the cry1Ac gene,9 MON 1445 the cp4 epsps gene10 and MON 15985 the cry1Ac and cry2Ab genes.11 Canola (Brassica napus L.) is mainly grown in the USA, Canada and India and used as a vegetable oil in food for humans and feed for livestock. Two events of biotech canola are typical: GT 73 and J Sci Food Agric (2014)
MS 8/RF 3. GT 73 contains the cp4 epsps and gox v247 genes12 and MS 8/RF 3 the bar gene.13 MS 8/RF 3 canola is a gene-stacked hybrid between MS 8 and RF 3.13 Soybean (Glycine max L.) is one of the world’s largest sources of plant protein and oil. One biotech soybean event is typical, MON 89788, which contains the cp4 epsps gene.14 Many consumers are concerned about the potential risk of biotech crops to human health and the ecological environment. Some countries have established labeling systems based on their own criteria, with thresholds for adventitious presence (AP) of biotech crops defined as 0.9% in the EU,15 3% in Korea16 and 5% in Japan.17 The detection method is very important to control the genetically modified organism (GMO) labeling system or living modified organism (LMO) management for biotech crops. The polymerase chain reaction (PCR) is the most efficient DNA-based detection method to identify biotech crops.18 Many PCR methods have been developed to detect the presence of biotech crops. Recently, the multiplex PCR19 – 22 and DNA chip23,24 have been developed as new methods. There are four types of analytical approaches: screening, gene-specific, construct-specific and event-specific analysis.25
∗
Correspondence to: Seong-Hun Lee, Component Analysis Division, Experiment Research Institute of National Agricultural Products Quality Management Service, Gimcheon, 740-871 Korea. E-mail: [email protected] / [email protected]
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© 2014 Society of Chemical Industry
www.soci.org The screening and gene-specific methods are applicable to detect many events of biotech plants using regulatory elements and trait genes. In this work a screening DNA chip was developed with 13 different element probes. Four plant probes were specific for soybean, maize, cotton and canola respectively. The probes of two regulatory elements and seven trait genes were specific for P35S, tNOS, epsps1, epsps2, cry1Ac, cry3B, pat, bar and pmi. The event-specific method has the highest specificity for one event by the junction region between the inserted DNA and the host genome of the biotech crop. In the case of maize, many biotech events have been produced, so an event-specific and efficient detection method for several biotech events simultaneously is needed. An event-specific multiplex PCR was developed to simultaneously detect five biotech maize events: MIR 604, Event 3272, LY 038, DAS-59122-7 and MON 88017. In this study the new analytical methods proved to have the ability to detect several targets on a DNA chip or agarose gel by one PCR at the same time, which could save on working time and load.
MATERIALS AND METHODS Materials Biotech maize NK 603, MON 863, MIR 604, DAS-59122-7 and Event 3272 seed powders were purchased from IRMM (Institute for Reference Materials and Measurements, Geel, Belgium). MON 88017 seed powder was purchased from AOCS (American Oil Chemists’ Society, Urbana, IL, USA). LY 038 seed powder was obtained from Monsanto Company (St. Louis, MO, USA). Biotech cotton MON 531, MON 1445 and MON 15985 seed powders were purchased from AOCS. Biotech canola MS 8, RF 3 leaf DNAs and GT 73 seed powder were purchased from AOCS. Biotech soybean MON 89788 seed powder was purchased from AOCS. DNA extraction Plant samples were ground in an electric mill (Pulverisette 14, Fritsch, Idar-Oberstein, Germany). Genomic DNA was extracted from 1 g of ground sample using a DNeasy Plant Maxi Kit (Qiagen, Hilden, Germany) according to the manufacturer’s manual, with slight modification. The incubation time was extended to 1 h after the addition of AP1 buffer and RNase A solution to the ground sample in the first extraction step, and genomic DNA was eluted with water incubated at 65 ∘ C. The quality of the extracted DNA was determined by UV spectrophotometry (ND-1000, NanoDrop Technologies, Wilmington, DE, USA) and confirmed by electrophoresis on agarose gel. Oligonucleotide primers and probes All primers and probes were designed using Primer Express 3.0 software (Applied Biosystems, Foster City, CA, USA). They were synthesized and purified by Genotech (Daejeon, Korea). The nucleotide sequences of event-specific multiplex PCR primers are listed in Table 1. The primers were designed by multiplex and event specificity for five biotech maize events.26 DNA Walking SpeedUp Premix Kits (Seegene, Seoul, Korea) were used to analyze the junction regions between the inserted DNAs and the host genomes of biotech crop events. M500-F/R primers were designed for detection of MIR 604 based on the 3′ junction region between the nopaline synthase (NOS) gene (GenBank Accession No. V00087) and the maize genome. M500-F and E135-R primers were designed for detection of Event 3272 based on the 3′ flanking region between the NOS gene and the maize genome.
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S-H Lee
L248-F/R primers were designed for detection of LY 038 based on the 3′ flanking region between the globulin 1 (Glb1) gene (GenBank Accession No. X59084) and the maize genome. M151-F/R primers were designed for detection of MON 88017 based on the 5′ junction region between the maize genome and the actin 1 promoter (GenBank Accession No. S44221). D81-F/R primers were designed for detection of DAS-59122-7 based on the 3′ flanking region between T-DNA and the maize genome from US Patent 2006/0070139. For the screening DNA chip the published four primer pairs were used as the reference genes for four crops: soybean, maize, cotton and canola.27 – 29 Nine primer pairs were designed for two regulatory elements and seven trait genes: P35S, tNOS, epsps1, epsps2, cry1Ac, cry3B, pat, bar and pmi. The nucleotide sequences of primers are listed in Table 2. Event-specific multiplex PCR for biotech maize events Multiplex PCR was performed to confirm the specificity of the designed event-specific primers for each of events MIR 604, Event 3272, LY 038, MON 88017 and DAS-59122-7. The sensitivity of multiplex PCR was also assessed using the DNA mixture from the five biotech maize events and non-biotech maize at the following levels: 0, 0.1, 0.5, 1 and 5% (w/w). Multiplex PCR was run using a GeneAmp PCR System 9700 (Applied Biosystems). A 30 𝜇L volume of reaction solution contained 80 ng of mixed genomic DNA, 1× PCR buffer, 0.2 mmol L−1 dNTPs, 1.5 mmol L−1 MgCl2 , 1.6 μmol L−1 M500-F primer, 0.8 μmol L−1 M500-R, L248-F, L248-R, D81-F and D81-R primer each, 0.4 μmol L−1 E135-R, M151-F and M151-R primer each and 1.04 units of AmpliTaqGold DNA polymerase (Applied Biosystems). The PCR was performed according to the following program: 1 cycle of 10 min at 94 ∘ C, 40 cycles of 30 s at 94 ∘ C, 1 min at 60 ∘ C and 1 min at 72 ∘ C, and a final extension cycle of 7 min at 72 ∘ C. Screening and gene-specific simplex PCR for DNA chip Simplex PCRs were performed to verify the regulatory elementand trait gene-specific PCR products as the probes of the DNA chip for four biotech crops’ 12 events using a GeneAmp PCR System 9700 (Applied Biosystems). A 25 𝜇L volume of reaction solution contained 50 ng of genomic DNA, 1× Taq-PCR mix (Genotech) and 0.5 μmol L−1 of each primer. The PCR was performed according to the following program: 1 cycle of 10 min at 94 ∘ C, 40 cycles of 30 s at 94 ∘ C, 30 s at 60 ∘ C and 30 s at 72 ∘ C, and a final extension cycle of 7 min at 72 ∘ C. Making of DNA chip and labeling with Syto61 Aminosilane-coated slides (Nuricell, Seoul, Korea) were used as solid supports to which PCR product probes were linked. The PCR product probes were mixed in Micro Spotting Solution Plus 2× (TeleChem, Sunnyvale, CA, USA) and transferred to a 384-well polystyrene plate (Genetix, Wokingham, UK) for printing. The probes were printed in duplicate on slides by a MicroGrid II (BioRobotics, Cambridge, UK). The printed slides were stored in containers overnight and the probes were immobilized by crosslinking at 200 mJ using a Bio-Link crosslinker (Vilber Lourmat, France). The crosslinked slides were stored in slide boxes at room temperature. Syto61 (Molecular Probes, Eugene, OR, USA) staining was performed as a quality control for the printed DNA chip slides. For the Syto61 solution, 5 μmol L−1 Syto61 was diluted in distilled water to a final dilution of 1:100 000 (v/v). The printed slides were incubated in Syto61 solution for 15 min with gentle agitation at
© 2014 Society of Chemical Industry
J Sci Food Agric (2014)
Detection of biotech crops by DNA chip and multiplex PCR methods
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Table 1. List of primers for event-specific multiplex PCR Target
Name
MIR 604
M500-F M500-R M500-F E135-R L248-F L248-R M151-F M151-R D81-F D81-R
Event 3272 LY 038 MON 88017 DAS-59122-7
Sequence (5′ → 3′ ) GCC GGT CTT GCG ATG ATT CCT GTG GTG GAG ATC TTT ATT TCG GCC GGT CTT GCG ATG ATT AGA TGT TTA TTT CAC ATG CAG ATG ACT CCC AGG TAG TAA TGC ACA GAT ATG C TTG AAT GCT CCA TTT GTA GTT CTT G CAG AGC GCT AAG CAG CAG AA CAT TGC TGA TCC ATG TAG ATT TCC TTA AAA ACG TCC GCA ATG TGT TA TTT AGA GGA TAA ACA AAC GGG ACC
Specificity
Length (bp)
tNOS maize genome tNOS maize genome Glb1 maize genome T-DNA maize genome T-DNA maize genome
500 341 248 151 81
Table 2. List of primers for screening DNA chip Gene
Target
Reference genes
Soybean Maize Cotton Canola
Regulatory elements
P35S tNOS
Trait genes
epsps1 epsps2 cry1Ac cry3B pat bar pmi
Sequence (5′ → 3′ )
Name Le1-F Le1-R SSIIb3-F SSIIb3-R fsACP-1 F fsACP-1R Hmg-1 F Hmg-1R P35S- F1 P35S-R1 tNOS-F1 tNOS-R1 EPSPS1-F EPSPS1-R EPSPS2-F EPSPS2-R cry1A-F cry1A-R cry3B-F cry3B-R pat-F pat-R bar-F bar-R pmi-F pmi-R
GCC CTC TAC TCC ACC CCC A GCC CAT CTG CAA GCC TTT TT CCA ATC CTT TGA CAT CTG CTC C GAT CAG CTT TGG GTC CGG A CAA ACA AGA GAC CGT GGA TAA GGT A CAA GAG AAT CAG CTC CAA GAT CAA G CCG TTT CAT ATG GTA GAT CTG ATT TTT T GAT CGC CGT CTT GTT GCA A GAT GTG ATA TCT CCA CTG ACG TAA GG CGT GTC CTC TCC AAA TGA AAT G CGT TCA AAC ATT TGG CAA TAA AG CGC TAT ATT TTG TTT TCT ATC GCG TAT TGC AGG TGA AAT CGG AAG AC TGC AGC ATC TTT TCC GTA TG TCC GTA TTC CAG GTG ACA AGT CTA CTT GCA TAG CCT TAC CAG TGT TGA GCC TGA GTT GTC CGT GAT CCC T CAG GCA CAA CCA GGA CGG AAC CCA CGG AGG TCC TGG ACA ACT C GAT CAG GAC TTC GAC TTG GGC C CGC AAG GTT TTA AGT CTG TGG TGG GTA ACT GGC CTA ACT GG GCT CCA CGC TCT ACA CCC ACC T GCT GCC AGA AAC CCA CGT CAT ACA GCC ACT CTC CAT TCA GG AGG GAG ACA ATC TCG GAA AA
room temperature. The slides were washed with gentle agitation in distilled water for 5 min, three times. The slides were spin-dried for 1 min at 1300 × g and scanned with a ScanArray 5000 (Packard BioChip Technologies, Billerica, MA, USA). Multiplex PCR with Cy5-dCTP labeling Multiplex PCR was performed using a GeneAmp PCR System 9700 (Applied Biosystems). A 20 𝜇L volume of reaction solution contained 40 ng of genomic DNA, 1× Taq-PCR mix (Genotech), 50 nmol L−1 Cy5-dCTP (GeneChem, Daejeon, Korea) and 0.5 μmol L−1 Le1-F/R, 0.5 μmol L−1 SSIIb3-F/R, 0.5 μmol L−1 fsACP-1 F/1R, 0.7 μmol L−1 Hmg-1 F/1R, 0.7 μmol L−1 EPSPS1-F/R, 0.5 μmol L−1 EPSPS2-F/R, 0.3 μmol L−1 cry1A-F/R, 0.3 μmol L−1 cry3B-F/R, 0.3 μmol L−1 pat-F/R, 0.5 μmol L−1 bar-F/R, 0.5 μmol L−1 J Sci Food Agric (2014)
Length (bp)
Reference 27
118 27
114 28
116 111
29
103
This work
200
This work
199
This work
129
This work
203
This work
197
This work
199
This work
189
This work
198
This work
pmi-F/R, 0.7 μmol L−1 tNOS-F1/R1 and 0.8 μmol L−1 P35S-F1/R1 primers. The PCR was performed according to the following program: 1 cycle of 10 min at 94 ∘ C, 40 cycles of 30 s at 94 ∘ C, 30 s at 60 ∘ C and 30 s at 72 ∘ C, and a final extension cycle of 7 min at 72 ∘ C. Hybridization and signal detection The printed slides were prehybridized at 42 ∘ C for 45 min in preheated buffer (5× sodium chloride/sodium citrate (SSC), 1 g L−1 sodium dodecyl sulfate (SDS), 10 g L−1 bovine serum albumin (BSA)). The purified Cy5-dCTP-labeled PCR products were mixed with 2× hybridization buffer (500 g L−1 formamide, 10× SSC, 2 g L−1 SDS), heat-denatured at 95 ∘ C for 5 min and hybridized at 42 ∘ C in a water bath in a sealed hybridization chamber (Genomictree,
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S-H Lee
Table 3. Analysis table for screening DNA chip 1
2
3
4
5
6
(bp)
Plant
Promoter Terminator
Trait gene
Event
500 341 248 151 81
(B) M
1
2
3
4
5
(bp) 500
Maize Maize Maize Maize Maize Cotton Cotton Canola Canola Soybean
P35S P35S P35S P35S – P35S P35S – – –
tNOS tNOS tNOS – tNOS tNOS tNOS tNOS – –
epsps1 NK 603 cry3Bb1 MON 863 epsps1/cry3Bb1MON 88017 pat DAS-59122-7 pmi MIR 604, Event 3272 cry1Ac MON 531, MON 15985 epsps2 MON 1445 bar MS 8/RF 3 epsps2 GT 73 epsps2 MON 89788
341 248 151
each crop: biotech maize NK 603, biotech cotton MON 531, biotech canola GT 73 and biotech soybean MON 89788.
81
Figure 1. Specificity and sensitivity of multiplex PCR primer pairs for five biotech maize events. PCR products were electrophoresed on 20 g L−1 agarose gel. Arrowheads indicate the expected PCR amplification products. The template DNAs of each lane were as follows. (A) Lanes 1–6: MIR 604, Event 3272, LY 038, MON 88017, DAS-59122-7 and mixed five biotech maize events respectively. (B) Lanes 1–5: 0, 0.1, 0.5, 1 and 5% (w/w) of mixed five biotech maize events. Lane M: 100 bp ladder.
Daejeon, Korea) overnight. After hybridization, to increase stringency, the slides were washed under agitation with the following solutions: once with solution 1 (1× SSC, 2 g L−1 SDS) for 3 min at hybridization temperature, once with solution 2 (0.1× SSC, 2 g L−1 SDS) for 3 min at room temperature and once with solution 3 (0.1× SSC) for 3 min at room temperature. The slides were then spin-dried at 1300 × g for 1 min and scanned and analyzed with a ScanArray 5000 (Packard BioChip Technologies). Quantitative analysis based on fluorescence intensities was also performed using QuatArray 2.0 software (Packard BioChip Technologies). Specificity and sensitivity of screening DNA chip The specificity test of the DNA chip was performed for four biotech crops’ 12 events: biotech maize NK 603, MON 863, MON 88017, MIR 604, Event 3272 and DAS-59122-7, biotech cotton MON 531, MON 1445 and MON 15985, biotech canola GT 73 and MS 8/RF 3 and biotech soybean MON 89788. The sensitivity test was performed using DNA from each event at the following levels; 0, 0.1, 0.5 and 1% (w/w). An in-house validation test was also performed using other samples that were selected as the representative event of
RESULTS Event-specific multiplex PCR The event-specific multiplex PCR was developed to simultaneously detect five biotech maize events: MIR 604, Event 3272, LY 038, MON 88017 and DAS-59122-7. The designed primer pairs were confirmed to be event-specific for the respective biotech maize events. The nine primers were also mixed and then amplified with the mixed template DNAs from 16 ng of each of the five biotech maize events. As a result, five amplified fragments were observed: 500 bp for MIR 604, 341 bp for Event 3272, 248 bp for LY 038, 151 bp for MON 88017 and 81 bp for DAS-59122-7. No amplification was observed from non-biotech maize (Fig. 1(A)). A sensitivity test was performed to determine the limit of detection (LOD) of the multiplex PCR method by three times repeat. The test DNA samples were mixtures of non-biotech maize and the five biotech maize events at the following levels: 0, 0.1, 0.5, 1 and 5% (w/w). As a result, the relative and absolute LODs for multiplex PCR were 0.5% and 0.4 ng respectively for the five biotech maize events (Fig. 1(B)). Screening DNA chip Thirteen PCR products were amplified from simplex PCRs by 13 primer pairs for four biotech crops’ 12 events (data not shown). The verified PCR products were used as the 13 probes in the DNA chip. The multiplex PCR was also performed using 13 primer pairs. However, it is difficult to distinguish PCR products of similar size
Soybean Soybean negative
Maize
Maize
Position epsps2
epsps2
epsps1
epsps1
cry1Ac
cry1Ac
pmi
cry3B
cry3B
pat
pat
pmi
bar
bar
P35S
P35S
Position
tNOS
tNOS
Cotton
Cotton negative Canola Canola
Figure 2. Microarray format of screening PCR product probes: right, microarray printing scheme for DNA chip; left, Syto61 staining image after printing microarray slide.
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© 2014 Society of Chemical Industry
J Sci Food Agric (2014)
Detection of biotech crops by DNA chip and multiplex PCR methods
www.soci.org (G) Cotton MON 531
(A) Maize NK 603 Soybean Soybean negative
Maize
Maize
Soybean Soybean negative
Maize
Maize
epsps1
epsps1
Position
epsps2
epsps2
epsps1
epsps1
Position
epsps2
epsps2
cry1Ac
cry1Ac
pmi
cry3B
cry3B
cry1Ac
cry1Ac
pmi
cry3B
cry3B
pat
pat
pmi
bar
bar
pat
pat
pmi
bar
bar
P35S
P35S
Position
tNOS
tNOS
P35S
P35S
Position
tNOS
tNOS
Cotton
Cotton
negative
Canola
Canola
Cotton
Cotton
negative
Canola
Canola
(B) Maize MON 863
(H) Cotton MON 15985 Soybean Soybean negative
Maize
Maize
Soybean Soybean negative
Maize
Maize
epsps1
epsps1
Position
epsps2
epsps2
epsps1
epsps1
Position
epsps2
epsps2
cry1Ac
cry1Ac
pmi
cry3B
cry3B
cry1Ac
cry1Ac
pmi
cry3B
cry3B
pat
pat
pmi
bar
bar
pat
pat
pmi
bar
bar
P35S
P35S
Position
tNOS
tNOS
P35S
P35S
Position
tNOS
tNOS
Cotton
Cotton
negative
Canola
Canola
Cotton
Cotton
negative
Canola
Canola
(C) Maize MON 88017
(I) Cotton MON 1445 Soybean Soybean negative
Maize
Maize
Soybean Soybean negative
Maize
Maize
epsps1
epsps2
epsps2
epsps1
epsps1
Position
epsps2
epsps2
cry1Ac
cry1Ac
pmi
cry3B
cry3B
epsps1
Position
cry1Ac
cry1Ac
pmi
cry3B
cry3B
pat
pat
pmi
bar
bar
pat
pat
pmi
bar
bar
P35S
Position
tNOS
tNOS
Cotton
negative Canola
Canola
P35S
P35S
Position
tNOS
tNOS
P35S
Cotton
Cotton
negative
Canola
Canola
Cotton
(D) Maize MIR 604
(J) Canola GT73 Soybean Soybean negative
Maize
Maize
Soybean Soybean negative
Maize
Maize
epsps1
epsps1
Position
epsps2
epsps2
epsps1
epsps1
Position
epsps2
epsps2
cry1Ac
cry1Ac
pmi
cry3B
cry3B
cry1Ac
cry1Ac
pmi
cry3B
cry3B
pat
pat
pmi
bar
bar
pat
pat
pmi
bar
bar
P35S
P35S
Position
tNOS
tNOS
P35S
P35S
Position
tNOS
tNOS
Cotton
Cotton
negative
Canola
Canola
Cotton
Cotton
negative
Canola
Canola
(E) Maize Event 3272
(K) Canola MS 8/RF 3 Soybean Soybean negative
Maize
Maize
Soybean Soybean negative
Maize
Maize
epsps1
epsps1
Position
epsps2
epsps2
epsps1
epsps1
Position
epsps2
epsps2
cry1Ac
cry1Ac
pmi
cry3B
cry3B
cry1Ac
cry1Ac
pmi
cry3B
cry3B
pat
pat
pmi
bar
bar
pat
pat
pmi
bar
bar
P35S
P35S
Position
tNOS
tNOS
P35S
P35S
Position
tNOS
tNOS
Cotton
Cotton
negative
Canola
Canola
Cotton
Cotton
negative
Canola
Canola
(F) Maize DAS-59122-7
(L) Soybean MON 89788 Soybean Soybean negative
Maize
Maize
Soybean Soybean negative
Maize
Maize
epsps1
epsps1
Position
epsps2
epsps2
epsps1
epsps1
Position
epsps2
epsps2
cry1Ac
cry1Ac
pmi
cry3B
cry3B
cry1Ac
cry1Ac
pmi
cry3B
cry3B
pat
pat
pmi
bar
bar
pat
pat
pmi
bar
bar
P35S
P35S
Position
tNOS
tNOS
P35S
P35S
Position
tNOS
tNOS
Cotton
Cotton
negative
Canola
Canola
Cotton
Cotton
negative
Canola
Canola
Figure 3. Hybridizaton of PCR product probes with multiplex PCR targets on DNA chip. Hybridization of PCR product probes and Cy5-labeled multiplex PCR target amplicons for four biotech crops’ 12 events.
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Table 4. Relative signal intensity values in screening DNA chip development
Item Soybean Maize Cotton Canola P35S tNOS Epsps1 Epsps2 Cry1Ac Cry3B Pat Bar Pmi Negative (spot solution) Position (Cy5-positive)
NK 603
MON 863
MON 88017
MIR 604
Event 3272
0.2 17.5 0.0 0.1 4.6 53.1 42.9 0.0 0.0 0.1 0.0 0.3 0.0 0.0
0.0 16.9 0.0 0.0 5.2 11.8 0.3 0.1 0.3 53.0 0.1 0.0 0.1 0.0
0.0 23.9 0.0 0.0 6.5 99.5 19.9 0.2 0.0 94.5 0.4 0.0 1.3 0.0
0.3 11.9 0.2 0.1 1.5 98.8 0.2 0.1 0.1 0.3 0.2 0.1 42.6 0.0
0.0 13.0 0.0 0.0 1.1 92.9 0.0 0.0 0.0 0.1 0.0 0.0 55.6 0.0
100.0
100.0
100.0
100.0
100.0
Table 5. Relative signal intensity values in screening DNA chip validation Item Soybean Maize Cotton Canola P35S tNOS Epsps1 Epsps2 Cry1Ac Cry3B Pat Bar Pmi Negative (spot solution) Position (Cy5-positive)
NK 603 0.0 20.1 0.0 0.0 5.1 51.3 29.4 0.0 0.0 0.1 0.0 0.0 0.0 0.0 100.0
MON 531 0.1 0.0 16.7 0.0 12.8 45.2 0.5 0.0 25.1 0.1 0.0 0.0 0.0 0.0 100.0
GT 73 0.0 0.0 0.0 60.2 0.2 0.2 0.0 64.5 0.1 0.0 0.0 0.0 0.0 0.0 100.0
MON 89788 29.9 0.2 0.2 0.0 0.3 0.1 0.0 25.8 0.8 0.9 0.5 0.5 0.2 0.0 100.0
on agarose gel, so we confirmed the results of multiplex PCR on the DNA chip containing 13 different probes. Four plant probes were specific for soybean, maize, cotton and canola respectively. Two regulatory element and seven gene probes were specific for P35S, tNOS, epsps1, epsps2, cry1Ac, cry3B, pat, bar and pmi (Fig. 2). The specificity of the DNA chip was confirmed by hybridization of the labeled multiplex PCR targets for four biotech crops’ 12 events (Table 3). In the case of maize, the target NK 603 (T-NK 603) hybridized to maize, epsps1, P35S and tNOS probes, T-MON 863 hybridized to maize, cry3B, P35S and tNOS probes, T-MON 88017 hybridized to maize, epsps1, cry3B, P35S and tNOS probes, T-MIR 604 and T-Event 3272 hybridized to maize, pmi and tNOS probes and T-DAS-59122-7 hybridized to maize, pat and P35S probes. In the case of cotton, T-MON 531 and T-MON 15985 hybridized to cotton, cry1Ac, P35S and tNOS probes and T-MON 1445 hybridized to cotton, epsps2, P35S and tNOS probes. In the case of canola, T-GT 73 hybridized to canola and epsps2 probes
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DAS59122-7
MON 531
MON 15985
MON 1445
GT 73
MS 8/ RF 3
MON 89788
0.3 38.2 0.0 0.0 13.2 1.0 0.4 0.0 0.0 0.0 93.8 0.3 0.2 0.0
0.1 0.0 50.3 0.0 27.8 98.8 0.7 0.0 87.1 0.1 0.0 0.0 0.0 0.0
0.0 0.0 35.1 0.0 28.7 90.3 0.6 0.0 87.2 0.0 0.0 0.0 0.1 0.0
0.0 0.0 18.7 0.0 10.9 57.5 0.2 26.1 0.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 55.9 0.5 0.0 0.1 60.1 0.1 0.0 0.0 0.0 0.5 0.0
0.0 0.0 0.0 28.9 0.4 79.1 0.0 0.0 0.0 0.5 0.0 26.7 0.0 0.0
25.7 0.4 0.0 0.0 0.4 0.0 0.0 26.1 0.0 0.0 0.1 0.0 0.0 0.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
and T-MS 8/RF 3 hybridized to canola, bar and tNOS probes. In the case of soybean, T-MON 89788 hybridized to soybean and epsps2 probes. The sensitivity of the DNA chip was confirmed to 0.5% level for all 12 events by the 13 primers. There were some false negatives of hybridization at 0.1% level (data not shown). The results of specificity and sensitivity at 0.5% are shown in Fig. 3. The results of the in-house validation test reconfirmed the specificity and 0.5% LOD for four crops’ four events. The results of hybridization were analyzed by spot colors or relative intensity values. The colors white, red, yellow, green, blue and black are revealed depending on the signal intensity. White indicates the strongest hybridization, while black indicates no hybridization. The colors of the hybridized positive spots appeared light blue to white, while those of the non-hybridized negative spots were dark blue to black (Fig. 3). On the other hand, for the relative intensity values, the mean value of the highest intensity from the Cy5-labeled position was set to 100%. The result was regarded as positive when the relative signal intensity value of the element from the position was more than 2%. This baseline was also mentioned in another paper.24 The relative intensity values of hybridization were 4.2–99.5%. The intensity of tNOS was the strongest and that of P35S the weakest. The relative intensity values of non-hybridization showed a maximum of 1.5% (i.e.
Revised: 12 February 2014
Accepted article published: 24 February 2014
Published online in Wiley Online Library:
(wileyonlinelibrary.com) DOI 10.1002/jsfa.6625
Screening DNA chip and event-specific multiplex PCR detection methods for biotech crops Seong-Hun Lee* Abstract BACKGROUND: There are about 80 biotech crop events that have been approved by safety assessment in Korea. They have been controlled by genetically modified organism (GMO) and living modified organism (LMO) labeling systems. The DNA-based detection method has been used as an efficient scientific management tool. Recently, the multiplex polymerase chain reaction (PCR) and DNA chip have been developed as simultaneous detection methods for several biotech crops’ events. RESULTS: The event-specific multiplex PCR method was developed to detect five biotech maize events: MIR604, Event 3272, LY 038, MON 88017 and DAS-59122-7. The specificity was confirmed and the sensitivity was 0.5%. The screening DNA chip was developed from four endogenous genes of soybean, maize, cotton and canola respectively along with two regulatory elements and seven genes: P35S, tNOS, pat, bar, epsps1, epsps2, pmi, cry1Ac and cry3B. The specificity was confirmed and the sensitivity was 0.5% for four crops’ 12 events: one soybean, six maize, three cotton and two canola events. CONCLUSION: The multiplex PCR and DNA chip can be available for screening, gene-specific and event-specific analysis of biotech crops as efficient detection methods by saving on workload and time. © 2014 Society of Chemical Industry Keywords: biotech crops; event-specific; multiplex PCR; screening; gene-specific; DNA chip
INTRODUCTION Since the introduction of the first genetically modified (GM) tomato, Flavr Savr™, in 1994, biotech crops have been increasingly developed and commercialized. Compared with conventional crops, biotech crops display insect resistance, herbicide tolerance or some other advantageous function. The global area for biotech crops has increased continuously from 1.7 million hectares in 1996 to 170.3 million hectares in 2012. The global adoption rates for principal biotech crops are as follows: soybean 81%, maize 35%, cotton 81% and canola 30%.1 Maize (Zea mays L.) is the most commercialized biotech crop and also the world’s third leading cereal crop after wheat and rice. It has been cultivated for human food, animal feed and biofuel. Seven events of biotech maize are typical: NK 603, MON 863, MIR 604, Event 3272, LY 038, MON 88017 and DAS-59122-7. NK 603 contains the cp4 epsps gene,2 MON 863 the cry3Bb1 gene,3 MIR 604 the mcry3A and pmi genes,4 Event 3272 the amy797E and pmi genes,5 LY 038 the cordapA gene,6 MON 88017 the cry3Bb1 and cp4 epsps genes7 and DAS-59122-7 the cry34Ab1, cry35Ab1 and pat genes.8 Cotton (Gossypium hirsutum L.) is an important economic crop used as a fiber and in cooking oil, snack foods and important protein concentrates for livestock. Three events of biotech cotton are typical: MON 531, MON 1445 and MON 15985. MON 531 contains the cry1Ac gene,9 MON 1445 the cp4 epsps gene10 and MON 15985 the cry1Ac and cry2Ab genes.11 Canola (Brassica napus L.) is mainly grown in the USA, Canada and India and used as a vegetable oil in food for humans and feed for livestock. Two events of biotech canola are typical: GT 73 and J Sci Food Agric (2014)
MS 8/RF 3. GT 73 contains the cp4 epsps and gox v247 genes12 and MS 8/RF 3 the bar gene.13 MS 8/RF 3 canola is a gene-stacked hybrid between MS 8 and RF 3.13 Soybean (Glycine max L.) is one of the world’s largest sources of plant protein and oil. One biotech soybean event is typical, MON 89788, which contains the cp4 epsps gene.14 Many consumers are concerned about the potential risk of biotech crops to human health and the ecological environment. Some countries have established labeling systems based on their own criteria, with thresholds for adventitious presence (AP) of biotech crops defined as 0.9% in the EU,15 3% in Korea16 and 5% in Japan.17 The detection method is very important to control the genetically modified organism (GMO) labeling system or living modified organism (LMO) management for biotech crops. The polymerase chain reaction (PCR) is the most efficient DNA-based detection method to identify biotech crops.18 Many PCR methods have been developed to detect the presence of biotech crops. Recently, the multiplex PCR19 – 22 and DNA chip23,24 have been developed as new methods. There are four types of analytical approaches: screening, gene-specific, construct-specific and event-specific analysis.25
∗
Correspondence to: Seong-Hun Lee, Component Analysis Division, Experiment Research Institute of National Agricultural Products Quality Management Service, Gimcheon, 740-871 Korea. E-mail: [email protected] / [email protected]
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© 2014 Society of Chemical Industry
www.soci.org The screening and gene-specific methods are applicable to detect many events of biotech plants using regulatory elements and trait genes. In this work a screening DNA chip was developed with 13 different element probes. Four plant probes were specific for soybean, maize, cotton and canola respectively. The probes of two regulatory elements and seven trait genes were specific for P35S, tNOS, epsps1, epsps2, cry1Ac, cry3B, pat, bar and pmi. The event-specific method has the highest specificity for one event by the junction region between the inserted DNA and the host genome of the biotech crop. In the case of maize, many biotech events have been produced, so an event-specific and efficient detection method for several biotech events simultaneously is needed. An event-specific multiplex PCR was developed to simultaneously detect five biotech maize events: MIR 604, Event 3272, LY 038, DAS-59122-7 and MON 88017. In this study the new analytical methods proved to have the ability to detect several targets on a DNA chip or agarose gel by one PCR at the same time, which could save on working time and load.
MATERIALS AND METHODS Materials Biotech maize NK 603, MON 863, MIR 604, DAS-59122-7 and Event 3272 seed powders were purchased from IRMM (Institute for Reference Materials and Measurements, Geel, Belgium). MON 88017 seed powder was purchased from AOCS (American Oil Chemists’ Society, Urbana, IL, USA). LY 038 seed powder was obtained from Monsanto Company (St. Louis, MO, USA). Biotech cotton MON 531, MON 1445 and MON 15985 seed powders were purchased from AOCS. Biotech canola MS 8, RF 3 leaf DNAs and GT 73 seed powder were purchased from AOCS. Biotech soybean MON 89788 seed powder was purchased from AOCS. DNA extraction Plant samples were ground in an electric mill (Pulverisette 14, Fritsch, Idar-Oberstein, Germany). Genomic DNA was extracted from 1 g of ground sample using a DNeasy Plant Maxi Kit (Qiagen, Hilden, Germany) according to the manufacturer’s manual, with slight modification. The incubation time was extended to 1 h after the addition of AP1 buffer and RNase A solution to the ground sample in the first extraction step, and genomic DNA was eluted with water incubated at 65 ∘ C. The quality of the extracted DNA was determined by UV spectrophotometry (ND-1000, NanoDrop Technologies, Wilmington, DE, USA) and confirmed by electrophoresis on agarose gel. Oligonucleotide primers and probes All primers and probes were designed using Primer Express 3.0 software (Applied Biosystems, Foster City, CA, USA). They were synthesized and purified by Genotech (Daejeon, Korea). The nucleotide sequences of event-specific multiplex PCR primers are listed in Table 1. The primers were designed by multiplex and event specificity for five biotech maize events.26 DNA Walking SpeedUp Premix Kits (Seegene, Seoul, Korea) were used to analyze the junction regions between the inserted DNAs and the host genomes of biotech crop events. M500-F/R primers were designed for detection of MIR 604 based on the 3′ junction region between the nopaline synthase (NOS) gene (GenBank Accession No. V00087) and the maize genome. M500-F and E135-R primers were designed for detection of Event 3272 based on the 3′ flanking region between the NOS gene and the maize genome.
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S-H Lee
L248-F/R primers were designed for detection of LY 038 based on the 3′ flanking region between the globulin 1 (Glb1) gene (GenBank Accession No. X59084) and the maize genome. M151-F/R primers were designed for detection of MON 88017 based on the 5′ junction region between the maize genome and the actin 1 promoter (GenBank Accession No. S44221). D81-F/R primers were designed for detection of DAS-59122-7 based on the 3′ flanking region between T-DNA and the maize genome from US Patent 2006/0070139. For the screening DNA chip the published four primer pairs were used as the reference genes for four crops: soybean, maize, cotton and canola.27 – 29 Nine primer pairs were designed for two regulatory elements and seven trait genes: P35S, tNOS, epsps1, epsps2, cry1Ac, cry3B, pat, bar and pmi. The nucleotide sequences of primers are listed in Table 2. Event-specific multiplex PCR for biotech maize events Multiplex PCR was performed to confirm the specificity of the designed event-specific primers for each of events MIR 604, Event 3272, LY 038, MON 88017 and DAS-59122-7. The sensitivity of multiplex PCR was also assessed using the DNA mixture from the five biotech maize events and non-biotech maize at the following levels: 0, 0.1, 0.5, 1 and 5% (w/w). Multiplex PCR was run using a GeneAmp PCR System 9700 (Applied Biosystems). A 30 𝜇L volume of reaction solution contained 80 ng of mixed genomic DNA, 1× PCR buffer, 0.2 mmol L−1 dNTPs, 1.5 mmol L−1 MgCl2 , 1.6 μmol L−1 M500-F primer, 0.8 μmol L−1 M500-R, L248-F, L248-R, D81-F and D81-R primer each, 0.4 μmol L−1 E135-R, M151-F and M151-R primer each and 1.04 units of AmpliTaqGold DNA polymerase (Applied Biosystems). The PCR was performed according to the following program: 1 cycle of 10 min at 94 ∘ C, 40 cycles of 30 s at 94 ∘ C, 1 min at 60 ∘ C and 1 min at 72 ∘ C, and a final extension cycle of 7 min at 72 ∘ C. Screening and gene-specific simplex PCR for DNA chip Simplex PCRs were performed to verify the regulatory elementand trait gene-specific PCR products as the probes of the DNA chip for four biotech crops’ 12 events using a GeneAmp PCR System 9700 (Applied Biosystems). A 25 𝜇L volume of reaction solution contained 50 ng of genomic DNA, 1× Taq-PCR mix (Genotech) and 0.5 μmol L−1 of each primer. The PCR was performed according to the following program: 1 cycle of 10 min at 94 ∘ C, 40 cycles of 30 s at 94 ∘ C, 30 s at 60 ∘ C and 30 s at 72 ∘ C, and a final extension cycle of 7 min at 72 ∘ C. Making of DNA chip and labeling with Syto61 Aminosilane-coated slides (Nuricell, Seoul, Korea) were used as solid supports to which PCR product probes were linked. The PCR product probes were mixed in Micro Spotting Solution Plus 2× (TeleChem, Sunnyvale, CA, USA) and transferred to a 384-well polystyrene plate (Genetix, Wokingham, UK) for printing. The probes were printed in duplicate on slides by a MicroGrid II (BioRobotics, Cambridge, UK). The printed slides were stored in containers overnight and the probes were immobilized by crosslinking at 200 mJ using a Bio-Link crosslinker (Vilber Lourmat, France). The crosslinked slides were stored in slide boxes at room temperature. Syto61 (Molecular Probes, Eugene, OR, USA) staining was performed as a quality control for the printed DNA chip slides. For the Syto61 solution, 5 μmol L−1 Syto61 was diluted in distilled water to a final dilution of 1:100 000 (v/v). The printed slides were incubated in Syto61 solution for 15 min with gentle agitation at
© 2014 Society of Chemical Industry
J Sci Food Agric (2014)
Detection of biotech crops by DNA chip and multiplex PCR methods
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Table 1. List of primers for event-specific multiplex PCR Target
Name
MIR 604
M500-F M500-R M500-F E135-R L248-F L248-R M151-F M151-R D81-F D81-R
Event 3272 LY 038 MON 88017 DAS-59122-7
Sequence (5′ → 3′ ) GCC GGT CTT GCG ATG ATT CCT GTG GTG GAG ATC TTT ATT TCG GCC GGT CTT GCG ATG ATT AGA TGT TTA TTT CAC ATG CAG ATG ACT CCC AGG TAG TAA TGC ACA GAT ATG C TTG AAT GCT CCA TTT GTA GTT CTT G CAG AGC GCT AAG CAG CAG AA CAT TGC TGA TCC ATG TAG ATT TCC TTA AAA ACG TCC GCA ATG TGT TA TTT AGA GGA TAA ACA AAC GGG ACC
Specificity
Length (bp)
tNOS maize genome tNOS maize genome Glb1 maize genome T-DNA maize genome T-DNA maize genome
500 341 248 151 81
Table 2. List of primers for screening DNA chip Gene
Target
Reference genes
Soybean Maize Cotton Canola
Regulatory elements
P35S tNOS
Trait genes
epsps1 epsps2 cry1Ac cry3B pat bar pmi
Sequence (5′ → 3′ )
Name Le1-F Le1-R SSIIb3-F SSIIb3-R fsACP-1 F fsACP-1R Hmg-1 F Hmg-1R P35S- F1 P35S-R1 tNOS-F1 tNOS-R1 EPSPS1-F EPSPS1-R EPSPS2-F EPSPS2-R cry1A-F cry1A-R cry3B-F cry3B-R pat-F pat-R bar-F bar-R pmi-F pmi-R
GCC CTC TAC TCC ACC CCC A GCC CAT CTG CAA GCC TTT TT CCA ATC CTT TGA CAT CTG CTC C GAT CAG CTT TGG GTC CGG A CAA ACA AGA GAC CGT GGA TAA GGT A CAA GAG AAT CAG CTC CAA GAT CAA G CCG TTT CAT ATG GTA GAT CTG ATT TTT T GAT CGC CGT CTT GTT GCA A GAT GTG ATA TCT CCA CTG ACG TAA GG CGT GTC CTC TCC AAA TGA AAT G CGT TCA AAC ATT TGG CAA TAA AG CGC TAT ATT TTG TTT TCT ATC GCG TAT TGC AGG TGA AAT CGG AAG AC TGC AGC ATC TTT TCC GTA TG TCC GTA TTC CAG GTG ACA AGT CTA CTT GCA TAG CCT TAC CAG TGT TGA GCC TGA GTT GTC CGT GAT CCC T CAG GCA CAA CCA GGA CGG AAC CCA CGG AGG TCC TGG ACA ACT C GAT CAG GAC TTC GAC TTG GGC C CGC AAG GTT TTA AGT CTG TGG TGG GTA ACT GGC CTA ACT GG GCT CCA CGC TCT ACA CCC ACC T GCT GCC AGA AAC CCA CGT CAT ACA GCC ACT CTC CAT TCA GG AGG GAG ACA ATC TCG GAA AA
room temperature. The slides were washed with gentle agitation in distilled water for 5 min, three times. The slides were spin-dried for 1 min at 1300 × g and scanned with a ScanArray 5000 (Packard BioChip Technologies, Billerica, MA, USA). Multiplex PCR with Cy5-dCTP labeling Multiplex PCR was performed using a GeneAmp PCR System 9700 (Applied Biosystems). A 20 𝜇L volume of reaction solution contained 40 ng of genomic DNA, 1× Taq-PCR mix (Genotech), 50 nmol L−1 Cy5-dCTP (GeneChem, Daejeon, Korea) and 0.5 μmol L−1 Le1-F/R, 0.5 μmol L−1 SSIIb3-F/R, 0.5 μmol L−1 fsACP-1 F/1R, 0.7 μmol L−1 Hmg-1 F/1R, 0.7 μmol L−1 EPSPS1-F/R, 0.5 μmol L−1 EPSPS2-F/R, 0.3 μmol L−1 cry1A-F/R, 0.3 μmol L−1 cry3B-F/R, 0.3 μmol L−1 pat-F/R, 0.5 μmol L−1 bar-F/R, 0.5 μmol L−1 J Sci Food Agric (2014)
Length (bp)
Reference 27
118 27
114 28
116 111
29
103
This work
200
This work
199
This work
129
This work
203
This work
197
This work
199
This work
189
This work
198
This work
pmi-F/R, 0.7 μmol L−1 tNOS-F1/R1 and 0.8 μmol L−1 P35S-F1/R1 primers. The PCR was performed according to the following program: 1 cycle of 10 min at 94 ∘ C, 40 cycles of 30 s at 94 ∘ C, 30 s at 60 ∘ C and 30 s at 72 ∘ C, and a final extension cycle of 7 min at 72 ∘ C. Hybridization and signal detection The printed slides were prehybridized at 42 ∘ C for 45 min in preheated buffer (5× sodium chloride/sodium citrate (SSC), 1 g L−1 sodium dodecyl sulfate (SDS), 10 g L−1 bovine serum albumin (BSA)). The purified Cy5-dCTP-labeled PCR products were mixed with 2× hybridization buffer (500 g L−1 formamide, 10× SSC, 2 g L−1 SDS), heat-denatured at 95 ∘ C for 5 min and hybridized at 42 ∘ C in a water bath in a sealed hybridization chamber (Genomictree,
© 2014 Society of Chemical Industry
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www.soci.org (A) M
S-H Lee
Table 3. Analysis table for screening DNA chip 1
2
3
4
5
6
(bp)
Plant
Promoter Terminator
Trait gene
Event
500 341 248 151 81
(B) M
1
2
3
4
5
(bp) 500
Maize Maize Maize Maize Maize Cotton Cotton Canola Canola Soybean
P35S P35S P35S P35S – P35S P35S – – –
tNOS tNOS tNOS – tNOS tNOS tNOS tNOS – –
epsps1 NK 603 cry3Bb1 MON 863 epsps1/cry3Bb1MON 88017 pat DAS-59122-7 pmi MIR 604, Event 3272 cry1Ac MON 531, MON 15985 epsps2 MON 1445 bar MS 8/RF 3 epsps2 GT 73 epsps2 MON 89788
341 248 151
each crop: biotech maize NK 603, biotech cotton MON 531, biotech canola GT 73 and biotech soybean MON 89788.
81
Figure 1. Specificity and sensitivity of multiplex PCR primer pairs for five biotech maize events. PCR products were electrophoresed on 20 g L−1 agarose gel. Arrowheads indicate the expected PCR amplification products. The template DNAs of each lane were as follows. (A) Lanes 1–6: MIR 604, Event 3272, LY 038, MON 88017, DAS-59122-7 and mixed five biotech maize events respectively. (B) Lanes 1–5: 0, 0.1, 0.5, 1 and 5% (w/w) of mixed five biotech maize events. Lane M: 100 bp ladder.
Daejeon, Korea) overnight. After hybridization, to increase stringency, the slides were washed under agitation with the following solutions: once with solution 1 (1× SSC, 2 g L−1 SDS) for 3 min at hybridization temperature, once with solution 2 (0.1× SSC, 2 g L−1 SDS) for 3 min at room temperature and once with solution 3 (0.1× SSC) for 3 min at room temperature. The slides were then spin-dried at 1300 × g for 1 min and scanned and analyzed with a ScanArray 5000 (Packard BioChip Technologies). Quantitative analysis based on fluorescence intensities was also performed using QuatArray 2.0 software (Packard BioChip Technologies). Specificity and sensitivity of screening DNA chip The specificity test of the DNA chip was performed for four biotech crops’ 12 events: biotech maize NK 603, MON 863, MON 88017, MIR 604, Event 3272 and DAS-59122-7, biotech cotton MON 531, MON 1445 and MON 15985, biotech canola GT 73 and MS 8/RF 3 and biotech soybean MON 89788. The sensitivity test was performed using DNA from each event at the following levels; 0, 0.1, 0.5 and 1% (w/w). An in-house validation test was also performed using other samples that were selected as the representative event of
RESULTS Event-specific multiplex PCR The event-specific multiplex PCR was developed to simultaneously detect five biotech maize events: MIR 604, Event 3272, LY 038, MON 88017 and DAS-59122-7. The designed primer pairs were confirmed to be event-specific for the respective biotech maize events. The nine primers were also mixed and then amplified with the mixed template DNAs from 16 ng of each of the five biotech maize events. As a result, five amplified fragments were observed: 500 bp for MIR 604, 341 bp for Event 3272, 248 bp for LY 038, 151 bp for MON 88017 and 81 bp for DAS-59122-7. No amplification was observed from non-biotech maize (Fig. 1(A)). A sensitivity test was performed to determine the limit of detection (LOD) of the multiplex PCR method by three times repeat. The test DNA samples were mixtures of non-biotech maize and the five biotech maize events at the following levels: 0, 0.1, 0.5, 1 and 5% (w/w). As a result, the relative and absolute LODs for multiplex PCR were 0.5% and 0.4 ng respectively for the five biotech maize events (Fig. 1(B)). Screening DNA chip Thirteen PCR products were amplified from simplex PCRs by 13 primer pairs for four biotech crops’ 12 events (data not shown). The verified PCR products were used as the 13 probes in the DNA chip. The multiplex PCR was also performed using 13 primer pairs. However, it is difficult to distinguish PCR products of similar size
Soybean Soybean negative
Maize
Maize
Position epsps2
epsps2
epsps1
epsps1
cry1Ac
cry1Ac
pmi
cry3B
cry3B
pat
pat
pmi
bar
bar
P35S
P35S
Position
tNOS
tNOS
Cotton
Cotton negative Canola Canola
Figure 2. Microarray format of screening PCR product probes: right, microarray printing scheme for DNA chip; left, Syto61 staining image after printing microarray slide.
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© 2014 Society of Chemical Industry
J Sci Food Agric (2014)
Detection of biotech crops by DNA chip and multiplex PCR methods
www.soci.org (G) Cotton MON 531
(A) Maize NK 603 Soybean Soybean negative
Maize
Maize
Soybean Soybean negative
Maize
Maize
epsps1
epsps1
Position
epsps2
epsps2
epsps1
epsps1
Position
epsps2
epsps2
cry1Ac
cry1Ac
pmi
cry3B
cry3B
cry1Ac
cry1Ac
pmi
cry3B
cry3B
pat
pat
pmi
bar
bar
pat
pat
pmi
bar
bar
P35S
P35S
Position
tNOS
tNOS
P35S
P35S
Position
tNOS
tNOS
Cotton
Cotton
negative
Canola
Canola
Cotton
Cotton
negative
Canola
Canola
(B) Maize MON 863
(H) Cotton MON 15985 Soybean Soybean negative
Maize
Maize
Soybean Soybean negative
Maize
Maize
epsps1
epsps1
Position
epsps2
epsps2
epsps1
epsps1
Position
epsps2
epsps2
cry1Ac
cry1Ac
pmi
cry3B
cry3B
cry1Ac
cry1Ac
pmi
cry3B
cry3B
pat
pat
pmi
bar
bar
pat
pat
pmi
bar
bar
P35S
P35S
Position
tNOS
tNOS
P35S
P35S
Position
tNOS
tNOS
Cotton
Cotton
negative
Canola
Canola
Cotton
Cotton
negative
Canola
Canola
(C) Maize MON 88017
(I) Cotton MON 1445 Soybean Soybean negative
Maize
Maize
Soybean Soybean negative
Maize
Maize
epsps1
epsps2
epsps2
epsps1
epsps1
Position
epsps2
epsps2
cry1Ac
cry1Ac
pmi
cry3B
cry3B
epsps1
Position
cry1Ac
cry1Ac
pmi
cry3B
cry3B
pat
pat
pmi
bar
bar
pat
pat
pmi
bar
bar
P35S
Position
tNOS
tNOS
Cotton
negative Canola
Canola
P35S
P35S
Position
tNOS
tNOS
P35S
Cotton
Cotton
negative
Canola
Canola
Cotton
(D) Maize MIR 604
(J) Canola GT73 Soybean Soybean negative
Maize
Maize
Soybean Soybean negative
Maize
Maize
epsps1
epsps1
Position
epsps2
epsps2
epsps1
epsps1
Position
epsps2
epsps2
cry1Ac
cry1Ac
pmi
cry3B
cry3B
cry1Ac
cry1Ac
pmi
cry3B
cry3B
pat
pat
pmi
bar
bar
pat
pat
pmi
bar
bar
P35S
P35S
Position
tNOS
tNOS
P35S
P35S
Position
tNOS
tNOS
Cotton
Cotton
negative
Canola
Canola
Cotton
Cotton
negative
Canola
Canola
(E) Maize Event 3272
(K) Canola MS 8/RF 3 Soybean Soybean negative
Maize
Maize
Soybean Soybean negative
Maize
Maize
epsps1
epsps1
Position
epsps2
epsps2
epsps1
epsps1
Position
epsps2
epsps2
cry1Ac
cry1Ac
pmi
cry3B
cry3B
cry1Ac
cry1Ac
pmi
cry3B
cry3B
pat
pat
pmi
bar
bar
pat
pat
pmi
bar
bar
P35S
P35S
Position
tNOS
tNOS
P35S
P35S
Position
tNOS
tNOS
Cotton
Cotton
negative
Canola
Canola
Cotton
Cotton
negative
Canola
Canola
(F) Maize DAS-59122-7
(L) Soybean MON 89788 Soybean Soybean negative
Maize
Maize
Soybean Soybean negative
Maize
Maize
epsps1
epsps1
Position
epsps2
epsps2
epsps1
epsps1
Position
epsps2
epsps2
cry1Ac
cry1Ac
pmi
cry3B
cry3B
cry1Ac
cry1Ac
pmi
cry3B
cry3B
pat
pat
pmi
bar
bar
pat
pat
pmi
bar
bar
P35S
P35S
Position
tNOS
tNOS
P35S
P35S
Position
tNOS
tNOS
Cotton
Cotton
negative
Canola
Canola
Cotton
Cotton
negative
Canola
Canola
Figure 3. Hybridizaton of PCR product probes with multiplex PCR targets on DNA chip. Hybridization of PCR product probes and Cy5-labeled multiplex PCR target amplicons for four biotech crops’ 12 events.
J Sci Food Agric (2014)
© 2014 Society of Chemical Industry
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S-H Lee
Table 4. Relative signal intensity values in screening DNA chip development
Item Soybean Maize Cotton Canola P35S tNOS Epsps1 Epsps2 Cry1Ac Cry3B Pat Bar Pmi Negative (spot solution) Position (Cy5-positive)
NK 603
MON 863
MON 88017
MIR 604
Event 3272
0.2 17.5 0.0 0.1 4.6 53.1 42.9 0.0 0.0 0.1 0.0 0.3 0.0 0.0
0.0 16.9 0.0 0.0 5.2 11.8 0.3 0.1 0.3 53.0 0.1 0.0 0.1 0.0
0.0 23.9 0.0 0.0 6.5 99.5 19.9 0.2 0.0 94.5 0.4 0.0 1.3 0.0
0.3 11.9 0.2 0.1 1.5 98.8 0.2 0.1 0.1 0.3 0.2 0.1 42.6 0.0
0.0 13.0 0.0 0.0 1.1 92.9 0.0 0.0 0.0 0.1 0.0 0.0 55.6 0.0
100.0
100.0
100.0
100.0
100.0
Table 5. Relative signal intensity values in screening DNA chip validation Item Soybean Maize Cotton Canola P35S tNOS Epsps1 Epsps2 Cry1Ac Cry3B Pat Bar Pmi Negative (spot solution) Position (Cy5-positive)
NK 603 0.0 20.1 0.0 0.0 5.1 51.3 29.4 0.0 0.0 0.1 0.0 0.0 0.0 0.0 100.0
MON 531 0.1 0.0 16.7 0.0 12.8 45.2 0.5 0.0 25.1 0.1 0.0 0.0 0.0 0.0 100.0
GT 73 0.0 0.0 0.0 60.2 0.2 0.2 0.0 64.5 0.1 0.0 0.0 0.0 0.0 0.0 100.0
MON 89788 29.9 0.2 0.2 0.0 0.3 0.1 0.0 25.8 0.8 0.9 0.5 0.5 0.2 0.0 100.0
on agarose gel, so we confirmed the results of multiplex PCR on the DNA chip containing 13 different probes. Four plant probes were specific for soybean, maize, cotton and canola respectively. Two regulatory element and seven gene probes were specific for P35S, tNOS, epsps1, epsps2, cry1Ac, cry3B, pat, bar and pmi (Fig. 2). The specificity of the DNA chip was confirmed by hybridization of the labeled multiplex PCR targets for four biotech crops’ 12 events (Table 3). In the case of maize, the target NK 603 (T-NK 603) hybridized to maize, epsps1, P35S and tNOS probes, T-MON 863 hybridized to maize, cry3B, P35S and tNOS probes, T-MON 88017 hybridized to maize, epsps1, cry3B, P35S and tNOS probes, T-MIR 604 and T-Event 3272 hybridized to maize, pmi and tNOS probes and T-DAS-59122-7 hybridized to maize, pat and P35S probes. In the case of cotton, T-MON 531 and T-MON 15985 hybridized to cotton, cry1Ac, P35S and tNOS probes and T-MON 1445 hybridized to cotton, epsps2, P35S and tNOS probes. In the case of canola, T-GT 73 hybridized to canola and epsps2 probes
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DAS59122-7
MON 531
MON 15985
MON 1445
GT 73
MS 8/ RF 3
MON 89788
0.3 38.2 0.0 0.0 13.2 1.0 0.4 0.0 0.0 0.0 93.8 0.3 0.2 0.0
0.1 0.0 50.3 0.0 27.8 98.8 0.7 0.0 87.1 0.1 0.0 0.0 0.0 0.0
0.0 0.0 35.1 0.0 28.7 90.3 0.6 0.0 87.2 0.0 0.0 0.0 0.1 0.0
0.0 0.0 18.7 0.0 10.9 57.5 0.2 26.1 0.0 0.0 0.0 0.0 0.0 0.0
0.0 0.0 0.0 55.9 0.5 0.0 0.1 60.1 0.1 0.0 0.0 0.0 0.5 0.0
0.0 0.0 0.0 28.9 0.4 79.1 0.0 0.0 0.0 0.5 0.0 26.7 0.0 0.0
25.7 0.4 0.0 0.0 0.4 0.0 0.0 26.1 0.0 0.0 0.1 0.0 0.0 0.0
100.0
100.0
100.0
100.0
100.0
100.0
100.0
and T-MS 8/RF 3 hybridized to canola, bar and tNOS probes. In the case of soybean, T-MON 89788 hybridized to soybean and epsps2 probes. The sensitivity of the DNA chip was confirmed to 0.5% level for all 12 events by the 13 primers. There were some false negatives of hybridization at 0.1% level (data not shown). The results of specificity and sensitivity at 0.5% are shown in Fig. 3. The results of the in-house validation test reconfirmed the specificity and 0.5% LOD for four crops’ four events. The results of hybridization were analyzed by spot colors or relative intensity values. The colors white, red, yellow, green, blue and black are revealed depending on the signal intensity. White indicates the strongest hybridization, while black indicates no hybridization. The colors of the hybridized positive spots appeared light blue to white, while those of the non-hybridized negative spots were dark blue to black (Fig. 3). On the other hand, for the relative intensity values, the mean value of the highest intensity from the Cy5-labeled position was set to 100%. The result was regarded as positive when the relative signal intensity value of the element from the position was more than 2%. This baseline was also mentioned in another paper.24 The relative intensity values of hybridization were 4.2–99.5%. The intensity of tNOS was the strongest and that of P35S the weakest. The relative intensity values of non-hybridization showed a maximum of 1.5% (i.e.
