Rapid Screening Detection of Genetically Modified Crops by Loop

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New Analytical Methods

Rapid Screening Detection of Genetically Modified Crops by LoopMediated Isothermal Amplification with a Lateral Flow Dipstick Reona Takabatake, Yukari Kagiya, Yasutaka Minegishi, Satoshi Futo, Keisuke Soga, Kosuke Nakamura, Kazunari Kondo, Junichi Mano, and Kazumi Kitta J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01765 • Publication Date (Web): 27 Jun 2018 Downloaded from http://pubs.acs.org on June 27, 2018

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Journal of Agricultural and Food Chemistry 1

Bullet points (1) statement of the problem addressed and originality of the approach

(1) To manage the increased number of GM events, we developed a novel loop-mediated isothermal amplification (LAMP)-based detection method using lateral flow dipstick chromatography for comprehensive GMO inspection.

(2) contribution of the work to create new knowledge in the field

(2) The combinatory approach of LAMP which is an isothermal reaction, easy-to-use lateral flow dipstick chromatography, and furthermore a multiplex detection system will open up the possibilities for DNA-based assays.

(3) relevance of the work to advance research and impact to the field of agricultural and food chemistry

(3) Because the developed method is rapid and cost-effective, the method will be

applicable for monitoring the validity of the food labeling system in many situations, including on-site inspection.

ACS Paragon Plus Environment


Research article

Title: Rapid Screening Detection of Genetically Modified Crops by Loop-Mediated Isothermal Amplification with a Lateral Flow Dipstick Reona Takabatake,† Yukari Kagiya,‡ Yasutaka Minegishi,§ Satoshi Futo,‡ Keisuke Soga,ǁ Kosuke Nakamura,ǁ Kazunari Kondo,ǁ Junichi Mano,† and Kazumi Kitta*,†

*

Corresponding author. Tel.: +81-29-838-7369; Fax: +81-29-838-7369; E-mail:

[email protected] †

Division of Analytical Science, Food Research Institute, National Agriculture and Food

Research Organization: 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8642, Japan ‡

FASMAC Co., Ltd.: 5-1-3 Midorigaoka, Atsugi, Kanagawa 243-0041, Japan

§

Nippon Gene Co., Ltd.: 1-5, Kandanishiki-cho, Chiyoda-ku, Tokyo 101-0054, Japan

ǁ

National Institute of Health Sciences: 3-25-26, Tonomachi, Kawasaki-ku,

Kawasaki-shi, Kanagawa 210-9501, Japan

Number of color figures: 4 Number of tables: 1 Number of supplementary materials: 4


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ABSTRACT

2 3

We developed a novel loop-mediated isothermal amplification (LAMP)-based

4

detection method using lateral flow dipstick chromatography for genetically modified

5

(GM) soybean and maize events. The single-stranded tag hybridization (STH) for

6

chromatography printed-array strip (C-PAS) system was used for detections targeting

7

the cauliflower mosaic virus 35S promoter, mannose-6-phosphate isomerase gene,

8

Pisum sativum ribulose 1, 5-bisphosphate carboxylase terminator, a common sequence

9

between the Cry1Ab and Cry1Ac genes, and a GA21-specific sequence. The STH

10

C-PAS system was applicable for multiplex analyses to perform simultaneous detections.

11

The limit of detection was 0.5% or less for each target. By using the developed method,

12

the LAMP amplification was visually detected. Moreover, the detection could be carried

13

out without any expensive instruments, even for the DNA amplification steps, by virtue

14

of the isothermal reaction. We demonstrated that the rapid and useful method developed

15

here would be applicable for screening GM crops.

16 17

Key words: Loop-mediated isothermal amplification (LAMP); Genetically

18

modified (GM); Dipstick DNA chromatography; Rapid; Multiplex analysis

19 20 21 22



23 24

INTRODUCTION The planted area of genetically modified (GM) crops has been expanding, and the

25

global area planted with GM crops reached 181.5 million hectares in 2016, up from 1.7

26

million hectares in 1996.1 However, some consumers are still expressing concerns about

27

the utilization of genetically modified organisms (GMOs). In response, many countries

28

and regions have passed laws requiring food labeling systems to indicate the presence of

29

authorized GM crops. Several counties and areas set a specified threshold level of GM

30

crops such as 0.9% in the European Union (EU), 1% in Australia and New Zealand, 3%

31

in Korea, and 5% in Japan, and mandate the labeling of foods containing equal to the

32

threshold level or more of GM crops.2 Under such conditions, not only the planted area,

33

but also the number of varieties of GM events have been continuously increased. In a

34

total of 40 countries, 26 GM crops and 392 GM events have been approved for use as

35

food or feed or for environmental release.1 To manage the increased number of GM

36

events, efficient screening detection methods for comprehensive GMO inspection are

37

required.

38

Polymerase chain reaction (PCR) has been used for various types of genetic testing, as

39

a gold standard method including GMO detection3-8 worldwide, but performing PCR

40

takes a relatively long time, and requires expensive instruments and reagents. Currently,

41

several dipstick DNA chromatography technologies have become available to detect

42

PCR products,9-12 including the single-stranded tag hybridization (STH) for

43

chromatography printed-array strip (C-PAS). In the STH C-PAS system, one primer is

44

labeled with a single-stranded specific tag sequence and the other primer is labeled with

45

a biotin molecule. After PCR amplification, the C-PAS, which is a paper

46

chromatography strip on which the complementary oligonucleotides of the tag


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sequences are printed, will be dipped into the reaction mixture. The PCR products are

48

then immobilized with single tag hybridization and visually detected with

49

streptavidin-biotin interaction using a streptavidin-coated blue-colored latex

50

microsphere. The STH C-PAS detection system has been applied for a crop cultivar

51

discrimination9 and diagnostic purposes.10, 12

52

Loop-mediated isothermal amplification (LAMP) is a rapid, highly specific, and

53

isothermal DNA amplification technique using DNA polymerase with high strand

54

displacement activity.13 Many GMO detection methods using the LAMP technique have

55

been developed and reported.14-18 To detect LAMP products, several techniques such as

56

visualization of the turbidity of magnesium pyrophosphate precipitation,19 use of

57

fluorescent dyes,20 and electrochemical detection21 have been utilized. In this study, to

58

pursue more rapid and inexpensive GM crops detection, we applied the STH C-PAS

59

system for LAMP product detection. In LAMP analyses, normally, a set of 4 or 6

60

primers are utilized. We used a set of 6 primers for each target, and we carefully chose 2

61

primers out of 6 primers for each target. And the selected two primers were labeled with

62

either the tag sequence(s) or biotin molecule(s). After LAMP reactions, amplification

63

products were detected with C-PAS as a blue line on the membrane. We also developed

64

multiplex analyses for simultaneous detection.

65 66

MATERIALS AND METHODS

67

Plant materials. As for soybean and maize seeds, RRS, MON89788, MON87701,

68

MON87705, MON87769, and MON810, MON863, MON88017, MON87460,

69

MON89034, and NK603 were kindly provided by Monsanto Company (St. Louis, MO).

70

3272, Bt11, Event176, GA21, MIR162 and MIR604 were kindly provided by Syngenta



71

Seeds AG (Basel, Switzerland). TC1507 and DAS59122 were kindly provided by

72

Pioneer Hi-Bred International (Johnston, IA). A2704-12 was kindly provided by its

73

developer and T25 was directly imported from the USA. QC9651 maize produced by

74

Quality Technology International, Inc. (Elgin, IL) was used as a non-GM maize control.

75

DNA extraction. Soybean and maize genomic DNAs were extracted with the GM

76

quicker (NIPPON GENE, Tokyo) according to the Japanese standard analytical

77

methods.2 The concentration and quality of the extracted DNA solutions were evaluated

78

by measuring ultraviolet absorbance with an ND-1000 spectrophotometer (NanoDrop

79

Technologies, Wilmington, DE). The concentration of genomic DNA solutions was

80

adjusted at 50 ng/µL, and 100 ng was used as a template for the LAMP analyses.

81

Sample preparation. We used DNA solution-based and weight-based mixing samples.

82

To prepare the DNA solution-based samples, genomic DNAs were extracted from each

83

GM soybean and maize event, and from non-GM soybean and maize and adjusted at a

84

concentration of 50 ng/µL, and the GM and non-GM DNA solutions were mixed as

85

volume ratios. Samples containing GM soybean genomic DNA at concentrations of 0,

86

0.03, 0.05, 0.1, 0.3 and 0.5% and GM maize at 0, 0.05, 0.1, 0.3 and 0.5% in the

87

respective non-GM DNA solutions were prepared. These DNA solution-based mixing

88

samples were used for the limit of detection evaluations. We also prepared seven GM

89

soybean mixing samples consisting of 0.5% RRS; 0.5% MON89788; 0.5% MON87701;

90

0.5% RRS and MON89788; 0.5% RRS and MON87701; 0.5% MON89788 and

91

MON87701; and 0.5% RRS, MON89788, and MON87701, and seven GM maize DNA

92

mixing samples consisting of 0.5% MON810; 0.5% MIR162; 0.5% GA21; 0.5%

93

MON810 and MIR162; 0.5% MON810 and GA21; 0.5% MIR162 and GA21; and 0.5%

94

MON810, MIR162, and GA21. These DNA solution-based mixing samples were used


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for the multiplex detection analyses. Meanwhile, the weight-based mixing samples were

96

used for direct LAMP analyses. Two GM soybean samples were previously prepared

97

consisting of i) 0.5% RRS and 0.5% MON87701,14 and ii) 0.5% RRS, 0.5%

98

MON89788 and 0.5% A2704-12,22 and three GM maize samples, consisting of iii) 0.5%

99

MON88017 and 0.5% 3272,14 iv) 0.5% MON810 and 0.5% GA2123 and v) 0.4% Bt11

100

and 0.2% GA21.24

101

LAMP assay. For the detection of each target sequence, a set of six primers consisting

102

of two outer primers (F3 and B3), two inner primers (FIP and BIP), and two loop

103

primers (LoopF and LoopB) was used. The oligonucleotide primers were synthesized

104

by FASMAC (Kanagawa, Japan).

105

LAMP reactions were performed as described previously.14 Briefly, the reactions

106

were conducted in 25-µL volumes using 2.0 µL of template DNA solution, 15 µL of

107

isothermal master mix (OptiGene, Horsham, UK), 1.0 mM Tris-HCl (pH 8.0), 0.1 mM

108

dithiothreitol, and primers at final concentrations of 0.2 µM for F3 and B3, 1.6 µM for

109

FIP and BIP, and 0.8 µM for LoopF and LoopB. For the detection of the cauliflower

110

mosaic virus 35S promoter (P35S), 3.2 µM instead of 1.6 µM of FIP and BIP was used.

111

Amplification was performed at 65℃ for 30 min.

112

For the sample direct LAMP analyses, GenCheck DNA Extraction Reagent

113

(FASMAC) was used. For sample preparation, 400 µl of lysis buffer was added to

114

ground samples of 40 mg maize or 20 mg soybeans, heated for 10 min at 100℃, and

115

chilled on ice. The samples were then centrifuged at 15,000 × g for 5 min, and the

116

resulting supernatants were directly used as templates for LAMP analyses.

117

LAMP products were separately detected with Genie II, which is a real-time

118

fluorometer (OptiGene), agarose gel electrophoresis, and dipstick DNA strips. For the


Journal of Agricultural and Food Chemistry

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Genie II detection, amplification conditions were set as described previously.14 For the

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electrophoresis detection, 5 µL of LAMP products were electrophoresed on 3.0%

121

agarose

122

Tris-acetate-ethylenediaminetetraacetic acid (TAE) buffer.

123

Signal detection by dipstick DNA chromatography. Dipstick DNA chromatography

124

strips (C-PAS) and reagents were obtained from TBA (Miyagi, Japan). For LAMP

125

amplifications, the 5’ ends of FIP or LoopF primers were tagged with a spacer and a tag

126

sequence (either A1, A2 or A3), and LoopB primers were biotinylated for each target.

127

The tagged primer sets are summarized in Table S1. For the detection of GM events

128

containing P35S, an F1 tag was used, and FIP and LoopF were labeled for GM soybean

129

and maize detection, respectively. Just before detection by C-PAS, the developing

130

solvent was prepared as follows. Ten microliters of the eluent containing 150 mM NaCl

131

was diluted with the same volume of sterilized distilled water, and 1 µL of

132

streptavidin-coated blue latex suspension was added (the eluent and the suspension were

133

supplied by TBA). After LAMP amplification at 65℃ for 30 min, 1 µL of LAMP

134

reaction products was added to the developing solvent. Subsequently, the C-PAS4, on

135

which four complementary tag sequences (A1, A2, A3 and A4) are linearly printed was

136

dipped into the mixture. Visible blue line(s) would appear within 10-15 min, and the

137

signal appearance was finally judged at 1 hour after dipping. The analyses were

138

repeated 10 times for each target and each sample, and a control sample with no

139

template was also subjected to the all primer sets under the same condition.

140

Multiplex detection by dipstick DNA chromatography. We used the STH C-PAS

141

system for multiplex detections consisting of three target sequences. For GM soybean

142

detection, P35S, Pisum sativum ribulose 1, 5-bisphosphate carboxylase E9 terminator

gel

supplemented

with

0.5

µg/mL

of


ethidium

bromide

in

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(tE9), and a sequence common to the Cry1Ab and Cry1Ac genes (Cry1Ab/Cry1Ac)

144

were used, and for GM maize detection, P35S, mannose-6-phosphate isomerase gene

145

(pmi) and a GA21 construct-specific sequence (GA21) were employed. For three

146

individual targets, three different tag sequences were assigned, such as A1 for P35S, A2

147

for tE9 or pmi, and A3 for Cry1Ab/Cry1Ac or GA21, respectively. For triplex LAMP

148

amplifications, three primer sets, meaning a total of 18 primers, were mixed. For the

149

detection of GM soybeans, the concentrations of primer sets were elaborately adjusted.

150

One-third concentrations (0.067 µM for F3 and B3, 1.07 µM for FIP and BIP, 0.27 µM

151

for LoopF and LoopB) of the standard concentrations for the P35S primer set, the

152

standard concentrations for the tE9 primer set, and quarter concentrations (0.05 µM for

153

F3 and B3, 0.4 µM for FIP and BIP, 0.2 µM for LoopF and LoopB) for the

154

Cry1Ab/Cry1Ac primer set were added. For the detection of GM maize, one-third

155

concentrations for the P35S primer set, half concentrations (0.1 µM for F3 and B3, 0.8

156

µM for FIP and BIP, 0.4 µM for LoopF and LoopB) for the pmi primer set, and the

157

standard concentrations for the GA21 primer set were added.

158 159

RESULTS AND DISCUSSION

160

Evaluation of the specificity of dipstick detection for LAMP products. We designed

161

several targets sequences for LAMP analyses, including P35S, tE9, Cry1Ab/Cry1Ac,

162

pmi, and GA21, which enabled the detection of 6 GM soybean and 15 GM maize

163

events.14 To pursue more cost-effective detection, we attempted to develop a novel

164

detection method using the STH C-PAS system for the LAMP products. We used the

165

C-PAS4 membrane, on which the complementary sequences of 4 tag sequences

166

consisting of A1, A2, A3 and A4 were separately printed. In this study, A1, A2 and A3



167

tag sequences were used. We used 6 primers, F3, B3, FIP, BIP, LoopF, and LoopB for

168

each LAMP analysis, and among these primers, the FIP, BIP, LoopF, and LoopB

169

primers are incorporated in LAMP products (Figure 1). To label the LAMP primers by

170

the tag sequence or biotin, there are theoretically four different combinations: FIP and

171

BIP, LoopF and LoopB, FIP and LoopB, or LoopF and BIP. As shown in Figure S1 (A),

172

in the case of P35S, non-specific signals appeared in the combination of FIP and BIP

173

and that of LoopF and BIP, for the A1 tag sequence and biotin, respectively, although it

174

was rather weak for the combination of FIP and BIP. Meanwhile, the results obtained

175

from Genie II and agarose gel electrophoresis showed no amplifications from non-GM

176

maize using any primer-tag and biotin combinations, meaning that non-specific signals

177

may be observed depending on the combination of primer set using the STH C-PAS

178

system. (Figure S1, B and C). We then used the primer set including the A1 labelled

179

LoopF or FIP, and the biotin labeled LoopB for P35S detection. To avoid non-specific

180

signal appearance, we deliberately evaluated which combination of tag sequence and

181

biotin-labeled primer set was suitable for the specific detection for other target

182

sequences (data not shown). The suitable primer combinations of tag sequence and

183

biotin are summarized in Table S1. For the detection of P35S, the A1 tag sequence was

184

used for both GM soybean and maize events, and FIP and LoopF were labeled with the

185

tag sequence for GM soybean and maize, respectively. The A2 tag sequence was used

186

for the detection of GM soybeans containing tE9 or GM maizes containing pmi, and

187

LoopF primers were labeled in both cases. The A3 tag sequence was used for the

188

detection of GM events containing Cry1Ab/Cry1Ac or GA21, and FIP primers were

189

labeled. To confirm the specificity for each primer set, we used three GM soybean

190

events consisting of RRS, MON89788, and MON87701, and three GM maize events


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consisting of MON810, MIR162, and GA21. The P35S region is contained in RRS and

192

MON810, and the tE9, Cry1Ab/Cry1Ac, pmi, and GA21 regions are contained in

193

MON89788, MON87701, MIR162, and GA21, respectively. The specific signals were

194

only detected in respective GM events containing the target sequences (Figure 2).

195

Limit of Detection (LOD) evaluations. The LODs were evaluated as the lowest

196

concentration at which all 10 replicates were positive.25 To analyze the LODs, we used

197

DNA solution mixing samples which were prepared by mixing genomic DNA solution

198

from individual GM events and non-GM soybean or maize. The determined LODs for

199

each target and each GM event are listed in Table 1, and representative results are

200

shown in Figure S2. In each, only single expected signals were appeared, and no

201

unexpected signals were observed from non-GM soybean or maize, or other GM events

202

without target sequences (data not shown). All of the LODs were equal to or less than

203

0.5% for GM events including 6 GM soybean events and 15 GM maize events. In

204

particular, the LODs for GM soybeans were lower than 0.1% except in the detection of

205

MON87705 and MON87769 targeting tE9. We had previously evaluated LODs of

206

LAMP-mediated screening detection methods for using a real time fluorometer, Genie

207

II (OptiGene, UK).14 The LODs for GM maize events in this study were equal to those

208

of the previous methods except for Bt11 targeting Cry1Ab/Ac and 3272 targeting pmi.

209

The LODs of the two GM maize events and all GM soybean events, targeting P35S, tE9,

210

and Cry1Ab/Ac, were lower than those of the previous methods. Among other our

211

previous reports using PCR, conventional PCR analyses showed that the LOD of RRS

212

targeting a construct specific region was 0.1%,26 and the LOD of MON810 targeting

213

P35S was 0.2%.24 We have also developed real-time PCR array method for

214

comprehensive GM detection.27 In the real-time PCR array, the LODs of RRS targeting



215

P35S was 0.05%, and MON 810 targeting P35S was 0.25%, and MON89788 targeting

216

tE9 was 0.05%. In this study, the LODs of RRS targeting P35S was 0.03%, MON810

217

targeting P35S was 0.3%, and MON89788 targeting tE9 was 0.05% (Table 1). From

218

these comparisons, it is difficult to determine which one is better in terms of sensitivity.

219

At least, the developed method is undoubtedly most rapid, inexpensive and easy to use.

220

Direct LAMP detection. We have developed a direct LAMP detection method to

221

simplify and shorten the process of sample preparation using crude cell lysates derived

222

from ground soybean or maize seed samples without any DNA extraction and

223

purification steps.14 In the previous study, GenCheck DNA Extraction Reagent

224

(GenCheck reagent) (FASMAC) enabled us to prepare samples in less than 20 minutes

225

in a few steps consisting of a heat treatment and centrifugation.14 Then, we applied the

226

GenCheck reagent for the developed STH C-PAS system-mediated LAMP detection.

227

Five GM mixing samples were used to evaluate the direct LAMP analyses (Table S2).

228

For the two soybean samples, 0.5% RRS in sample i) and 0.5% RRS and 0.5%

229

A2704-12 in sample ii) were used for P35S detection, 0.5% MON89788 in sample ii)

230

was used for tE9 detection and 0.5% MON87701 in sample i) was used for

231

Cry1Ab/Cry1Ac detection. In maize samples, 0.5% MON88017 in sample iii), 0.5%

232

MON810 in sample iv), and 0.4% Bt11 in sample v) were used for P35S detection,

233

0.5% 3272 in sample iii) was used for pmi detection, and 0.5% and 0.2% GA21 in

234

samples iv) and v) were used for GA21 detection. Representative results are shown in

235

Figure 3. The blue line signals detecting P35S at the A1 position appeared in samples i),

236

ii), iii), iv) and v). Likewise, the signals only appeared in samples ii) and iii) at the A2

237

position, indicating that sample ii) was positive for tE9, and sample iii) was positive for

238

pmi detection. The signal appeared in samples i) and v) at the position for


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Cry1Ab/Cry1Ac, and in samples iv) and v) at the A3 position in GA21, indicating that

240

sample i) and v) were positive for Cry1Ab/Cry1Ac, and sample iv) and v) were positive

241

for GA21 detection. These results suggested that direct LAMP analysis was applicable

242

to the STH C-PAS detection system.

243

Multiplex detection by the STH C-PAS system. We used the three targets for the

244

screening detection of GM soybean and maize events consisting of P35S, tE9, and

245

Cry1Ac, and of P35S, pmi, and GA21, respectively. To enhance the simplicity and

246

rapidity of the process, we attempted to develop multiplex LAMP detection using the

247

STH C-PAS system. When three primer sets for each target sequence are mixed and

248

function successfully, different LAMP products can be simultaneously detected.

249

However, as shown in Table1, the detection sensitivities were not exactly at the same

250

level among the primer sets. In the case of GM soybean target sequences, the P35S

251

primer set (LOD ≤ 0.03%) showed higher sensitivity than the tE9 primer set (LOD ≤

252

0.05 or 0.3%). To normalize the signal detection sensitivities, we adjusted the primer set

253

concentrations of three targets for both GM soybean and maize events (data not shown).

254

Finally, we found that the ratio of 1/3 : 1 : 1/4 concentrations for the primer sets of P35S,

255

tE9 and Cry1Ac for the GM soybean detection, and the ratio of 1/3 : 1/2 : 1

256

concentrations for the primer sets of P35S, pmi and GA21 for the GM maize detection

257

were most effective. To evaluate the multiplex detection system, seven GM soybean

258

samples which contained one, two or three GM events including 0.5% RRS,

259

MON89788, and MON87701, and seven GM maize samples which contained one, two,

260

or three GM events including 0.5% MON810, MIR162, and GA21, were prepared as

261

DNA solution mixing samples. These obtained signal patterns of all samples were

262

completely consistent with expectations, indicating that multiplex detections could be



263

applicable for the LAMP detection (Figure 4). The LODs of multiplex detections were

264

evaluated in a similar manner of single detections, and 0.5% amount of two or three

265

different targets were simultaneously detected. Generally, for LAMP product detection,

266

electrophoreses,

267

precipitation19 or fluorescent dyes14 are widely utilized. However, these techniques have

268

not been applied to simultaneous multiple-target detection methods, because it is

269

impossible to distinguish LAMP amplifications derived from each target sequence

270

individually. In the developed STH C-PAS detection system, when each tag sequence

271

was assigned to a LAMP target sequence, the LAMP amplifications could be easily

272

identified by the position(s) of the signal(s) on the C-PAS membrane. We used the

273

C-PAS4 membrane on which complementary sequences of 4 tag sequences (A1, A2, A3

274

and A4) are printed. In this study, we used 3 tag sequences for both GM soybean and

275

maize, thus, one more sequence corresponding to A4 can be used if the need arises.

visualization

of

the

turbidity

of

magnesium pyrophosphate

276

We developed a novel LAMP-mediated screening detection method for GM soybean

277

and maize using dipstick DNA chromatography, namely, the STH C-PAS system.

278

Although many PCR-mediated detection methods using dipstick DNA chromatography

279

have already been reported,9-12 there are some disadvantages to PCR-mediated DNA

280

chromatography. For instance, fine temperature regulation is required for PCR reactions

281

consisting of denaturation, annealing, and extension steps, and for this, expensive

282

instruments are required. On the other hand, LAMP is an isothermal reaction, and

283

less-expensive instruments such as a water bath or an isothermal heating block could be

284

applicable. In many PCR-related detection methods, uracil-N glycosylase (UNG)

285

system has been utilized.28 In the UNG system, deoxyuridine triphosphate (dUTP)

286

instead of deoxythimine triphosphate (dTTP) is used for PCR amplification, and prior to


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subsequent PCR reactions, the reaction mixtures are pretreated with UNG which

288

specifically digests uracil-containing DNA. The UNG is then heat-inactivated just

289

before PCR amplification. It might be useful to apply UNG system to the developed

290

C-PAS mediated detection method to decrease the risk of carry over contamination,

291

although it does not seem easy because the DNA polymerase used for LAMP

292

amplification is heat-labile in general. We also developed a multiplex detection system

293

for LAMP products, which is more time- and cost-effective. The STH C-PAS detection

294

system for LAMP products could be used not only for GMO detection but also for other

295

genetic testing processes such as diagnostic microbial detections.

296

The developed method was demonstrated to be rapid and cost-effective, and the

297

LODs of the methods were at the same level as or lower than those in our previously

298

reported qualitative detection method including LAMP analyses.14, 24, 26 The LODs of

299

the developed methods were equal or less than 0.5% for all targets, indicating that the

300

developed methods are available not only for Japan but also other countries or areas

301

such as the EU, Australia, New Zealand, Korea in terms of the level of GM content.

302

Therefore, we conclude that the developed method would be applicable for monitoring

303

the validity of the food labeling system in many situations, including on-site inspection.

304 305

ASSOCIATED CONTENT

306

Supporting Information

307

Specificity test of different combinations of LAMP primers which labeled with tag

308

sequence and biotin (Figure S1). Representative result of LOD evaluations (Figure S2).

309

LAMP primers information used in this study (Table S1). Content of the weight-based

310

mixing samples (Table S2).



311 312

AUTHOR INFORMATION

313

Corresponding Author *Telephone: +81-29-838-736. Fax: +81-29-838-7369. E-mail: [email protected]

314 315

ORCID iD Reona Takabatake: 0000-0001-6419-73114

316

Kazumi Kitta: 0000-0002-3691-271X

317

Funding

318

This work was supported by the Ministry of Agriculture, Forestry, and Fisheries of

319

the Japan Research Project “Genomics-based Technology for Agricultural Improvement

320

GRA-201”.

321

Notes

322

The authors no competing financial interest.

323 324

ABBREVIATIONS USED

325

dTTP, deoxythimine triphosphate; dUTP, deoxyuridine triphosphate; GM, genetically

326

modified; GMO, genetically modified organism; LAMP, loop-mediated isothermal

327

amplification; LOD, limit of detection; P35S, cauliflower mosaic virus 35S promoter;

328

PCR, polymerase chain reaction; pmi, mannose-6-phosphate isomerase gene; STH

329

C-PAS, single-tag hybridization chromatographic printed array strip; tE9, E9 terminator;

330

UNG, uracil-N glycosylase.

331

REFERENCES

332

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333

Biotech/GM Crops: 2016; ISAAA Brief 52-2016; International Service for the

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Bioanal. Chem. 2003, 375, 985-993.

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(5) ISO24276:2006. Foodstuffs: Methods of analysis for the detection of genetically

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Koiwa, T.; Akiyama, H.; Teshima, R.; Futo, S.; Minegishi, Y. Interlaboratory

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study of qualitative PCR methods for genetically modified maize events MON810,

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Bt11, GA21, and CaMV P35S. J. AOAC Int. 2013, 96, 346-352.

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421 422

(28) Aslanzadeh, J. Preventing PCR amplification carryover contamination in a clinical laboratory. Ann. Clin. Lab. Sci. 2004, 34, 389-396.


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Page 21 of 31


423

F3

F2

B1c

Genome DNA

LoopB

LoopF F1c

B2

Tag sequence Tag sequence

Biotin

LAMP amplification

The Table of Contents （TOC）graphic


Detection by C-PAS


Page 22 of 31

22

(A)

(B)

F3

F2

B1c

Genome DNA

LoopB

LoopF F1c

B2

F3 primer FIP primer Tag sequence

B3 B3 primer

BIP primer LoopB primer

LoopF primer Tag sequence

Biotin

FIP or LoopF primer is tagged with Tag sequence

LAMP amplification Flow direction

C-PAS membrane

Figure 1. Schematic diagram for the detection of LAMP products using dipstick DNA chromatography (C-PAS). (A) LAMP amplification is performed using six primers consisting of F3, B3, FIP, BIP, LoopF and LoopB. The FIP primer consists of the 3’ end of an F2 region and the 5’ end of an F1c region, and the BIP primer consists of the 3’ end of a B2 region and the 5’ end of a B1c region. FIP or LoopF primers were labeled with a tag sequence, and the LoopB primer was labeled with biotin. (B) The complementary oligonucleotides of the tag sequences are preliminarily printed on the C-PAS membrane and are indicated by arrow heads. The resulting LAMP products are trapped by single-strand tag hybridization.


Page 23 of 31


(A)

1 2 3 4

1 2 3 4

1 2 3 4

5 6 7 8

5 6 7 8

5 6 7 8

P35S soy

tE9

Cry1Ac

P35S maize

pmi

GA21

Positional marker

A3 A2 A1

Positional marker

P35S soy

40k

2

20k

1, 3 ,4

0

-10k

0

5

10

15

20

25

30

80k

40k 20k

1, 2 ,4

-20k

10k

5, 7 ,8

-10k

0

5

10

15

80k

Cry1Ac

60k

20

25

30

4

40k 20k

1, 2 ,3

0

-20k

0

Fluorescence

Fluorescence

6

0

GA21

5

10

15

20

25

30

0

5

Time (min)

P35S maize

20k

3

0

5 6 7 8

pmi

tE9

60k

5 6 7 8

P35S maize

Time (min) 40k 30k

5 6 7 8

1 2 3 4 Cry1Ac

Fluorescence

50k

1 2 3 4 tE9

Fluorescence

Fluorescence

(C)

P35S soy

30k

7

pmi

20k 10k 0

5, 6 ,8

-10k

0

10

15

20

15

20

25

30

60k

25

30

8

GA21

40k 20k

5, 6 ,7

0

-20k

5

10

Time (min)

40k

Fluorescence

1 2 3 4

(B)

0

5

10

15

20

25

30

Figure 2. Specificity test for LAMP analyses for GM soybean and maize events. Representative results are shown with C-PAS (A), agarose gel electrophoresis (B), and Genie II (C). Lanes 1, 2, 3, 4, 5, 6, 7 and 8 are non-GM soybean, RRS, MON89788, MON87701, non-GM maize, MON810, MIR162, and GA21, respectively. The A1, A2, and A3 tag sequences were used for P35S, tE9, or pmi and Cry1Ab/Cry1Ac or GA21 detections, respectively.



Page 24 of 31

24

1 2 3 4 5 6 7

1 2 3 4 5 6 7

1 2 3 4 5 6 7

P35S soy

tE9

Cry1Ac

1 2 3 4 5 6 7

1 2 3 4 5 6 7

1 2 3 4 5 6 7

P35S maize

pmi

GA21

A3 (Cry1Ac) A2 (tE9) A1 (P35S)

A3 (GA21) A2 (pmi) A1 (P35S)

Figure 3. Direct LAMP detections with the STH C-PAS system. Representative results are shown with GM soybean and maize weight-based mixing samples. Lanes 1-3 show the results from soybean samples. Lane 1: sample i) 0.5% RRS and MON87701; lane 2: sample ii) 0.5% RRS, MON89788, and A2704-12; and lane 3: non-GM soybean. Lanes 4-7 show the results from maize samples. Lane 4: sample iii) 0.5% MON88017 and 3272; lane 5: sample iv) 0.5% MON810 and GA21; lane 6: sample v) 0.4% Bt11 and 0.2% GA21; and lane 7: non-GM maize. The upper panel was detected with primer sets designed for soy, in which the A1, A2 and A3 tag sequences were used for P35S, tE9


Page 25 of 31


and Cry1Ab/Cry1Ac detections, respectively. And the lower panel was detected with primer sets for maize, in which the A1, A2 and A3 corresponded to P35S, pmi and GA21, respectively.



1 2

3

4 5 6

9 10 11 12 13 14 15 16

7 8

soy

A3 (Cry1Ac) A2 (tE9) A1 (P35S)

maize

A3 (GA21) A2 (pmi) A1 (P35S)

Figure 4. Multiplex detection for LAMP products with the STH C-PAS system. Lanes 1-8 show the results from soybean samples. Lane 1: non-GM soybean; lane 2: 0.5% RRS; lane 3: 0.5% MON89788; lane 4: 0.5% MON87701; lane 5: 0.5% RRS and MON89788; lane 6: 0.5% RRS and MON87701; lane 7: 0.5% MON89788 and MON87701; lane 8: 0.5% RRS, MON89788, and MON87701. The A1, A2 and A3 tag sequences were used for P35S, tE9 and Cry1Ab/Cry1Ac detections, respectively. Lanes 9-16 show the results from maize samples. Lane 9: non-GM maize; lane 10: 0.5% MON810; lane 11: 0.5% MIR162; lane 12: 0.5% GA21; lane 13: 0.5% MON810 and MIR162; lane 14: 0.5% MON810 and GA21; lane 15: 0.5% MIR162 and GA21; lane 16: 0.5% MON810, MIR162, and GA21. The A1, A2, and A3 tag sequences were used for P35S, pmi and GA21 detections, respectively.


Page 26 of 31

Page 27 of 31


GM NonGM 1 2 3 4 1 2 3 4

(B)

50k

Fluorescence

(A)

P35S maize

P35S maize

80k

60k

40k

0

6

12

18

24

1 2 4 3

GM

3 4 2 1

NonGM

30

Time (min)

(C)

A1

1

2

3

4

(D) 1

2

3

4

A1

FIP

LoopF

FIP

LoopF

Biotin

BIP

LoopB

LoopB

BIP

Figure S1. Specificity evaluation for LAMP analyses. Representative results targeting P35S are shown for GM maize MON810 detection with C-PAS (A), Genie II (B), and agarose gel electrophoresis (C). Four primer sets containing A1 tag- or biotin-labeled primers were used for LAMP amplification and the combinations of tag primers were different for each lane. The combination of A1-tagged and biotinylated primers are summarized (D). In lanes 1, 2, 3, and 4, the A1 tag and biotin were labeled with FIP and BIP, LoopF and LoopB, FIP and LoopB, and LoopF and BIP, respectively.



Page 28 of 31

28

P35S

tE9

Cry1Ab/c

pmi

Figure S2. The LOD evaluation of the STH C-PAS-mediated detection for LAMP products. Representative results are shown for GM soybean and maize detection.


Page 29 of 31


Table 1 Summary of LOD evaluations for individual assays GMO amount, %

GMO amount, % Positive/Total

Target

GM event

P35S

RRS A2704-12

0.05% 0.05%

10 / 10 10 / 10

0.03% 0.03%

10 / 10 10 / 10

Bt11 Event176 MON810 T25 NK603 MON863 TC1507 DAS59122 MON88017 MON89034 MON87460

0.1% 0.5% 0.3% 0.3% 0.3% 0.3% 0.3% 0.3% 0.3% 0.5% 0.5%

10 / 10 10 / 10 10 / 10 10 / 10 10 / 10 10 / 10 10 / 10 10 / 10 10 / 10 10 / 10 10 / 10

0.05% 0.3% 0.1% 0.1% 0.1% 0.1% 0.1% 0.1% 0.1% 0.3% 0.3%

9 / 10 6 / 10 2 / 10 3 / 10 8 / 10 8 / 10 5 / 10 9 / 10 9 / 10 9 / 10 9 / 10

tE9

MON89788 MON87705 MON87769

0.1% 0.5% 0.5%

10 / 10 10 / 10 10 / 10

0.05% 0.3% 0.3%

10 / 10 10 / 10 10 / 10

Cry1Ab/ Cry1Ac

MON87701

0.1%

10 / 10

0.05%

10 / 10

Bt11

0.3%

10 / 10

0.1%

10 / 10

MIR604 MIR162 3272

0.3% 0.3% 0.3%

10 / 10 10 / 10 10 / 10

0.1% 0.1% 0.1%

6 / 10 6 / 10 6 / 10

GA21

0.1%

10 / 10

0.05%

9 / 10

pmi

GA21

Positive/Total



Table S1 The LAMP primers used in this study Target

Sequence

P35S (soybean)

F3 B3 FIP BIP LoopF LoopB

ATTGCGATAAAGGAAAGGCTATCG ACTTCCTTATATAGAGGAAGGGTC A1-GAAGACGTGGTTGGAACGTCTTCTTAGTGGTCCCAAAGATGGA GCAAGTGGATTGATGTGATATCTCCTTGCGAAGGATAGTGGGA TTTCCACGATGCTCCTCG Biotin-CGTAAGGGATGACGCACA

P35S (maize)


ATTGCGATAAAGGAAAGGCTATCG ACTTCCTTATATAGAGGAAGGGTC GAAGACGTGGTTGGAACGTCTTCTTAGTGGTCCCAAAGATGGA GCAAGTGGATTGATGTGATATCTCCTTGCGAAGGATAGTGGGA A1-TTTCCACGATGCTCCTCG Biotin-CGTAAGGGATGACGCACA

tE9


ACACCAGAATCCTACTGAGT GAATCTGACAAGGATTCTGGAA CCATCCATTTCCATTTCACAGTTCGTGAGTATTATGGCATTGGGA AAATGTGTCAAATCGTGGCCTCTAGCCTAGTGAATAAGCATAATGG A2-CAAGCACAACAAATGGTACAAG Biotin-TGACCGAAGTTAATATGAGGAG

Cry1Ab/Cry1Ac


TGATGGACATCTTGAACAGC CATAGGCGAACTCTGTTCC A3-CGCTGAATCCAACTGGAGAGGTCTACACCGATGCTCACA AACGCCGCTCCACAACAAGGAAGACAAGGTTCTGTAGAC GTGTCCAGACCAGTAATACTCTC Biotin-TATCGTTGCTCAACTAGGTCAG

pmi


CAGTTCACGAGTGCAGAAT CGGCTTGTGGTTAGGATC GAAAGGCAGTTCGCCAAAGCCGTGATGTGATTGAGAGTGATA CAGCACAGCCACTCTCCATTTGGCAAAACCGATTTCAGA A2-TCTCCGAGCAGAGTCGAT Biotin-CAGGTTCATCCAAACAAACACA

GA21


GGACTACTGCATCATCACG TGATAATCATCGCAAGACCG A3-CGGCAAGGGAGAAAGCCATGAGAAGCTGAACGTGACG GCTGAGCACTTTCGTCAAGAATTAAGTGCCAAATGTTTGAACGATC CATCCTGTGGTCGTCGTAC Biotin-CTCTAGAAGAAGCTTCGACGAA


Page 30 of 31

Page 31 of 31


Table S2 Contents of the weight-based mixing samples Target

P35S

tE9

Cry1Ab/Cry1Ac

Soybean

i) RRS(0.5%)+ MON87701(0.5%)

ii) RRS(0.5%)+ A2704-12(0.5%)+ MON89788(0.5)%

i) RRS(0.5%)+ MON87701(0.5%)

pmi

GA21

iii) MON88017(0.5%) +3272(0.5%)

iv) MON810(0.5%)+ GA21(0.5%)

ii) RRS(0.5%)+ A2704-12(0.5%)+ MON89788(0.5)%

Maize

iii) MON88017(0.5%) +3272(0.5%)

v) Bt11(0.4%)+ GA21(0.2%)

iv) MON810(0.5%)+ GA21(0.5%)

v) Bt11(0.4%)+ GA21(0.2%)

v) Bt11(0.4%)+ GA21(0.2%)

Underlined GM events contain each target segment. i)-v) correspond to the sample numbers in the text.


Rapid Screening Detection of Genetically Modified Crops by Loop

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