One Novel Multiple-Target Plasmid Reference Molecule Targeting

Subscriber access provided by UNIVERSITY OF ADELAIDE LIBRARIES

Article

One novel multiple-target plasmid reference molecule targeting eight genetically modified canola events for GM canola detection Zhuqing Li, Xiang Li, Canhua Wang, Guiwen Song, Liqun Pi, Lan Zheng, Dabing Zhang, and Litao Yang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b02453 • Publication Date (Web): 28 Aug 2017 Downloaded from http://pubs.acs.org on August 30, 2017

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 44

Journal of Agricultural and Food Chemistry

1

One novel multiple-target plasmid reference molecule

2

targeting eight genetically modified canola events for GM

3

canola detection

4 5

Zhuqing Li1,†, Xiang Li2,†, Canhua Wang1, Guiwen Song3, Liqun Pi1, Lan Zheng1, Dabing

6

Zhang1,4, Litao Yang1*.

7 8

1

9

Organisms, School of Life Sciences and Biotechnology, Shanghai Jiao Tong University,

National Center for the Molecular Characterization of Genetically Modified

10

Shanghai 200240, China.

11

2

12

1208 Minsheng Road, Shanghai 200135, P. R. China

13

3

14

Republic of China. Beijing 100025, China.

15

4

16

Collaborative Innovation Center of Regional Modern Agriculture and Environmental

17

Protection, Huaian 223300, China

Shanghai Entry-Exit Inspection and Quarantine Bureau, GMO Detection Laboratory,

Development Center of Science and Technology, Ministry of Agriculture of People’s

Key Laboratory of Crop Marker-Assisted Breeding of Huaian Municipality, Jiangsu

18 19

†These two authors contributed equally to this work.

20

*

21

34207174; Email: [email protected].

To whom correspondence should be addressed: Tel.: +86 21 34205073; Fax: +86 21

22 1

ACS Paragon Plus Environment


Page 2 of 44

23

Abstract

24

Multiple-target plasmid DNA reference materials have been generated and utilized as

25

good substitutes of matrix-based reference materials in the analysis of genetically

26

modified organisms (GMOs). Herein, we report the construction of one

27

multiple-target plasmid reference molecule, pCAN, which harbors eight GM canola

28

event-specific sequences (RF1, RF2, MS1, MS8, Topas 19/2, Oxy235, RT73, and T45)

29

and a partial sequence of the canola endogenous reference gene PEP. The

30

applicability of this plasmid reference material in qualitative and quantitative PCR

31

assays of the eight GM canola events was evaluated, including the analysis of

32

specificity, limit of detection (LOD), limit of quantification (LOQ), and performance of

33

pCAN in the analysis of various canola samples, etc. The LODs are 15 copies for RF2,

34

MS1, and RT73 assays using pCAN as the calibrator, and 10 genome copies for the

35

other events. The LOQ in each event-specific real-time PCR assay is 20 copies. In

36

quantitative real-time PCR analysis, the PCR efficiencies of all event-specific and PEP

37

assays are between 91% and 97 %, and the squared regression coefficients (R2) are all

38

higher than 0.99. The quantification bias values varied from 0.47% to 20.68% with

39

relative standard deviation (RSD) from 1.06% to 24.61% in the quantification of

40

simulated samples. Furthermore, 10 practical canola samples sampled from

41

imported shipments in the port of Shanghai, China were analyzed employing pCAN

42

as the calibrator, and the results were comparable with those assays using

43

commercial certified materials as calibrator. Concluding from these results, we

44

believe that this newly developed pCAN plasmid is one good candidate for being a 2


Page 3 of 44


45

plasmid DNA reference material in the detection and quantification of the eight GM

46

canola events in routine analysis.

47

Keywords: Multiple-target reference plasmid; Genetically Modified canola;

48

Qualitative PCR; Quantitative real-time PCR.

49

3



Page 4 of 44

50

Introduction

51

Canola (Brassica napus L.), one of the staple oil-crops, is a significant source of edible

52

oil and plant proteins in the world. It is mainly planted in China, India, and Canada

53

and holds an important status in the international agricultural product trade. GM

54

canola is the fourth commercialized GM crops. In 2016, the planted area of GM

55

canola has reached about 8.6 million hectares in global area, such as USA, Canada,

56

and Argentina, etc. 1 To date, a total of 43 GM Argentine canola and Polish canola

57

events, including the gene stacked events, have been approved for commercialization

58

and planting. In China, seven GM canola lines (MS1×RF1, MS8×RF3, MS1×RF2, RT73,

59

Oxy235, T45, and Topas 19/2) including nine GM events have been approved for

60

usage as raw and processed materials since 2004.2 China now imports more than one

61

million tons of canola each year, and 60 percent are GM canola. According to the

62

GMO labeling regulation issued by Chinese government in 2001, products derived

63

from GM canola should be labeled with a zero threshold. 3 To implement the labeling

64

regulation, pre-validated event-specific detection methods and reference materials

65

for each GM line need to be well developed. Currently, several techniques have been

66

developed for GMO analysis in the past decade, such as PCR 4-5, real-time PCR 6,

67

digital PCR 7-8, microarray 9, loop-mediated isothermal amplification (LAMP) 10-11, and

68

next generation sequencing 12. Regardless of which detection method to choose,

69

reference materials (RMs) which are used as calibrator are essential for quality

70

insurance to deliver accurate GM contents.

71

Reference materials play key roles in the development and evaluation of new 4


Page 5 of 44


72

methods, calibration of instruments, and routine analysis in many fields, such as

73

clinical diagnosis, food safety testing, chemical analysis, and GMO detection, etc. 13 In

74

GMO analysis, matrix-based RMs, genomic DNA RMs, and plasmid DNA RMs are the

75

mainly used RMs. Among them, matrix-based RMs and genomic DNA RMs have the

76

advantage of being most closely to practical samples. However, the production of

77

matrix-based or genomic DNA RM was limited by the availability of GM materials

78

with high purity and clear genotype. 2 Currently, commercial matrix-based and

79

genomic DNA certified reference materials (CRMs) are mainly produced by Reference

80

Materials and Measurements, European Commission (IRMM-EU) and the American

81

Oil Chemists’ Society (AOCS).

82

In 2002, Kuribara et al., developed plasmid reference molecules for GM soybean and

83

maize detection, and validated their applicability in GM soybean and maize

84

quantification through a collaborative ring trial. 14 Since then, more and more

85

plasmid reference molecules have begun to be used as calibrator in GMO detection.

86

The advantages and disadvantages of plasmid reference molecules have been well

87

discussed in several previous publications 14-17. Their main advantages are: i) plasmid

88

reference molecules can be easily constructed and produced with high quantity and

89

quality; ii) multiple GM sequences from different GM events can be recombined into

90

one plasmid, and one plasmid can be used as calibrator for multiple GM events; iii)

91

the production cost is quite low. The disadvantages include easy contamination,

92

limitation of specific DNA fragments and corresponding methods, and different

93

amplification efficiency in PCR reactions compared with using genomic DNA. Despite 5



Page 6 of 44

94

of these disadvantages, plasmid reference molecules have been widely accepted in

95

GMO analysis. Up to date, several publications have reported the development and

96

application of new plasmid reference molecules in GMO analysis. For example, Yang

97

et al. developed a multiple-target plasmid reference molecule for quantification of

98

nine GM maize events. 18 Zhang et al. constructed a plasmid reference material for

99

multiplex quantitative analysis of GM soybean GTS 40-3-2. 19 Lievens et al. validated

100

pJANUS-02-001 as a valid plasmid calibrator for GM soybean GTS 40-3-2. 20 Meng et

101

al. developed and validated a plasmid reference material for GM maize TC1507. 21

102

Kim et al. developed a simple plasmid reference for GM wheat MON71800. 22 Pi et al.

103

reported a plasmid reference material targeting five GM soybean events. 23 Also,

104

IRMM-EU developed and certified four plasmid DNA CRMs for GM maize events

105

MON810 (ERM-AD413), NK603 (ERM-AD415), 356043 (ERM- AD425), and 98140

106

(ERM-AD427).

107

For GM canola detection, the qualitative PCR 24-25, real-time PCR 26, and Microarray 27

108

methods have been developed and validated employing different GM canola RMs.

109

Up to date, a total of nine CRMs have been developed by AOCS and are commercially

110

available. All of the nine CRMs are matrix-based CRM or genomic DNA CRM,

111

including one seed CRM for event GT73/RT73, one seed powder CRM of MON88302,

112

and seven leaf genomic DNA CRMs for GM events MS1, Rf1, Rf2, Rf3, Topas 19/2, T45,

113

and MS8. However, few plasmid CRM or multiple-target CRM for GM canola events

114

detection were developed except for GT73 and Oxy235 events. 24, 28 In this study, we

115

developed one multiple-target plasmid CRM, pCAN, which contains the 6


Page 7 of 44


116

event-specific DNA fragments of eight GM canola events and a partial sequence of

117

canola endogenous reference gene PEP. The applicability of pCAN as a calibrator in

118

qualitative PCR and quantitative real-time PCR analysis of GM canola events were

119

also evaluated.

7



Page 8 of 44

120

Material and Methods

121

Materials and DNA Extraction

122

Reference materials of GM maize NK603 (ERM-BF415f) were purchased from

123

Institute for Reference Materials and Measurements (IRMM). Leaf tissue DNA or

124

seed powder of GM canola event RT73 (0304-B2), Ms1 (0711-A2), Rf1 (0711-B2), Rf2

125

(0711-C2), Topas 19/2 (0711-D3), T45 (0208-A5), Ms8 (0306-F6) and Rf3 (0306-G5)

126

were purchased from American Oil Chemists’ Society (AOCS). Seeds or leaves of GM

127

canola event Oxy235 and GM rice TT51-1 were kindly provided by the developers.

128

Non-GM canola seeds were purchased from a local market in Shanghai, China and

129

validated to be free of GM contents in our laboratory. Ten practical canola seed

130

samples were sampled from the port of Shanghai, China and supplied by Shanghai

131

Entry-exit Inspection and Quarantine Bureau. A Plant DNA Mini-Prep Kit (Ruifeng

132

Agro-tech Co. Ltd., Shanghai, China) was used to extract and purify genomic DNA

133

after the plant materials were ground in liquid nitrogen. A Plasmid Mini Kit (Watson

134

Biotechnologies, Inc., Shanghai, China) was used for isolating and purifying plasmid

135

DNA. The concentration and purity of each extracted genomic DNA or plasmid DNA

136

was evaluated according to the absorbance at 230nm, 260nm and 280 nm

137

determined using a NanoDrop 1000 spectrophotometer (NanoDrop Technologies,

138

LLC, Wilmington, DE, USA). The integrity of extracted genomic DNAs and plasmid

139

DNAs were further checked by 0.8% agarose gel electrophoresis. The copy number in

140

each genomic DNA or plasmid DNA sample was calculated according to the

141

determined DNA quantity and genome size 29. 8


Page 9 of 44


142

Oligonucleotide Primers and Probes

143

Primers for cloning the event-specific DNA fragments were designed based on the

144

event-specific sequence of each event using Primer Premier 5.0 software (PREMIER

145

Biosoft International, CA). Primers for qualitative PCR analysis were adopted from

146

Chinese national standards for GM canola detection. Primers and probes for

147

quantitative real-time PCR assays of the eight events were designed using Beacon

148

Designer 7.2 software (PREMIER Biosoft International, Palo Alto,CA, USA). For GM

149

canola events RF1, RF2, MS1, MS8, Oxy235 and T45, the 5' end flanking sequence

150

were used as target fragments. For GM canola events Topas 19/2 and GT73/RT73,

151

the 3'-end event-specific sequence was amplified in real-time PCR analysis. The

152

primer pair of Q-PEP-1F/2R and probe Q-PEP-P were designed and used to amplify

153

canola endogenous reference gene PEP. The primers and probes for real-time PCR

154

assays were listed in Table 1. All primers and probes were synthesized by Invitrogen

155

Co., Ltd. (shanghai China).

156

Construction of the multiple-target plasmid pCAN

157

To construct the multiple-target pCAN by molecular cloning, the pEASY-T3 Cloning

158

Vector (Beijing TransGen Biotech Co., Ltd. China) was selected as the basic vector,

159

and nine DNA fragments (eight event-specific sequences of RF1, RF2, MS1, MS8,

160

Topas19/2, Oxy235, RT73, and T45 as well as the partial sequence of canola PEP gene)

161

were inserted into the pEASY-T3 vector by conventional molecular cloning

162

techniques, such as overlapping PCR, restriction enzyme digestion and ligation. The

163

sequences of eight canola events and PEP gene were from NCBI database, including 9



Page 10 of 44

164

RF1 with Accession number EU090199.1, RF2 with EU090197.1, MS1 with

165

EU090198.1, MS8 with EU020107.1, Topas 19/2 with EU124676.1, Oxy235 with

166

EU099578.1, RT73 with HJ231561.1, T45 with FJ154954.1 and PEP gene with

167

D13987.1.

168

The eight event-specific DNA fragments and PEP gene fragment were first amplified

169

individually from templates by conventional PCR, and the corresponding primers are

170

listed in Supplementary Table S1. Next, three integrated DNA fragments of

171

MS1-RT73 named F1 (MS1+RT73), F2 (Oxy235+ MS8), and F3 (T45+Topas+RF2+RF1)

172

were obtained by overlapping PCR. Firstly, fragment F1 was cloned into the pEASY-T3

173

vector by T-A ligation, then fragment F2 was integrated by endogenous restriction

174

enzyme Pst I and Bgl II, and finally fragment F3 was ligated after enzyme Bgl II and

175

Xho I digestion. The sequence of the new integrated vector was confirmed by

176

sequencing. Then the new big pEASY-T3 vector was digested by enzyme Bgl II and

177

Xba I, and then ligated with the PEP gene fragment after digestion by enzyme BamH1

178

and Sal I. The finally assembled pCAN was confirmed twice by DNA sequencing

179

(Shanghai Meiji Biological Science and Technology Co. Ltd, Shanghai, China).

180

Qualitative PCR assays

181

Qualitative PCR assays were performed to evaluate the specificity and sensitivity

182

when pCAN was used as templates in PCR reactions. All PCR reactions were carried

183

out using a Verity Thermal Cycler (Applied Biosystems, Carlsbad, CA, USA) in a 25 μl

184

reaction volume, including 1×PCR buffer, 100 nM dNTP, 200 nM of each primer, 1.5

185

units of Taq DNA polymerase (TaKaRa Biotechnology Co., Ltd.,Dalian, China), 2 μl of 10


Page 11 of 44


186

DNA template, and ddH2O. The PCR amplification program was: 95°C for 7 min; 35

187

cycles of 95 °C for 30 s, 58 °C for 30 s, 72 °C for 30 s; a final extension step of 72 °C for

188

7 min. PCR products were examined by 2% agarose gel electrophoresis with Gelred

189

staining. Each PCR test was repeated three times and each time with three parallel

190

replicates.

191

Quantitative real-time PCR assays

192

The real-time PCR reactions were carried out using a Prism ABI 7900 detection

193

system (Applied Biosystems, Carlsbad, CA, USA) in a reaction volume of 25 μL, which

194

contained 1 × TaqMan Universal PCR Master Mix (Applied Biosystems, Carlsbad, CA,

195

USA), 400 nM of each primer, 200nM of the probe, 5 μL DNA template, and ddH2O to

196

fill up the final volume. The Real-time PCR program was: pre-denaturation at 95 °C

197

for 10 min, followed by 45 cycles of 95 °C for 15 s and 60 °C for 60 s. The fluorescent

198

signals were monitored and analyzed in the extension step using the Sequence

199

Detector Software (SDS) V2.4. Each real-time PCR reaction was repeated three times,

200

and each time with three parallel reactions.

201

Quantification of GM canola content using pCAN as a calibrator

202

The performance of pCAN as a calibrator in real-time PCR analysis was evaluated

203

using three mixed DNA samples and ten practical canola samples. Since the

204

discrepancy of PCR efficiency between using plasmid and genomic DNA templates

205

might influence the ratio of exogenous to endogenous genome copy number, a

206

conversion factor (Cf) is often used. 14, 21-23, 28 In general, a Cf value is defined as the

207

genome copy number ratio of the event-specific DNA to endogenous reference gene 11



Page 12 of 44

208

in a homogenous genomic DNA sample using the plasmid DNA CRM as a calibrator,

209

and calculated using the formula Cf =

210

determine the Cf values of pCAN, homogenous genomic DNAs of each GM event

211

were prepared at three concentrations (10.0, 5.0, 1.0 ng/μl). The Cf value for each

212

event is defined as the average of three Cf values determined in the three DNAs at

213

different concentrations. For Cf determination, each real-time PCR reaction was

214

repeated three times, and each time with three parallel replicates. After the Cf values

215

were determined, the GM content in each canola sample was calculated using the

216

formula GM amount (%) = × +.

. To

' × )**

217

12


Page 13 of 44


218

Results and Discussion

219

Construction of the reference Plasmid pCAN

220

We have developed a novel reference plasmid pCAN containing eight event-specific

221

GM canola DNA sequences and one canola endogenous reference gene PEP (Figure

222

1). The length of pCAN is 7972 bp including 3253 bp of the eight event-specific GM

223

canola and endogenous PEP sequences. The full length pCAN DNA sequence was

224

determined by DNA sequencing and the sequences of the eight event-specific GM

225

canola and endogenous PEP sequences were confirmed. The confirmed sequences of

226

the inserted GM event and PEP DNA fragments are same with our design and

227

expectation (As shown in Supplementary File 1).

228

Qualitative PCR assays using pCAN as calibrator

229

Specificity. For a given GM event, an event-specific DNA sequence is a unique

230

sequence specific to that GM event, so the specificity of an event-specific PCR assay

231

should be quite high. For a multiple-target reference plasmid, however, since

232

multiple event-specific DNA sequences are within close proximity in the same vector,

233

cross interferences might happen and unexpected amplicons might be produced in

234

an event-specific PCR assay using the reference plasmid as template. Therefore, the

235

specificity of using pCAN in each event-specific PCR assay should be tested

236

individually. We performed qualitative PCR assays of eight GM canola events (RF1,

237

RF2, MS1, MS8, Topas 19/2, Oxy235, RT73, and T45) as well as endogenous gene PEP,

238

and the specificity of each PCR assay when using pCAN as the template was

239

evaluated. In all nine PCR assays, only expected amplicons corresponding to their 13



Page 14 of 44

240

specific targets were obtained, and no unexpected amplicon was observed (Figure 2).

241

For instance, a 200 bp amplicon was expected and observed in RF1 event PCR assay

242

when pCAN plasmid and RF1 genomic DNA were used as templates, and no other

243

amplified DNA fragment was observed. The same held true for PCR assays of the

244

other GM canola events (RF2, MS1, MS8, Topas 19/2, Oxy235, RT73, and T45), as well

245

as assays of non-GM canola, GM maize NK603, GM rice TT51-1, and GM soybean

246

MON89788 (Figure 2A and 2J). In PEP assay, the expected 248 bp PEP DNA fragment

247

was specifically amplified using pCAN and each GM canola genomic DNAs as

248

template; no PEP amplification was observed when GM maize, soybean, and rice

249

genomic DNAs were used as template (Figure 2I and 2J). Also, the high specificity

250

was confirmed using the real-time PCR assays with Sybr Green I dyer (As shown in

251

supplementary Figure S1). These results indicate that pCAN has high specificity for

252

each of the nine targets contained in the plasmid, and it could be used as calibrator

253

in PCR assays of the eight GM canola events.

254

Sensitivity. The sensitivities of using pCAN as calibrator in the nine PCR assays were

255

evaluated using serially diluted pCAN DNA solutions. These serially diluted pCAN

256

DNA solutions had concentrations of 5000, 1000, 250, 125, 100, 50, 25, 15, and 10

257

copies per reaction, respectively. The test results showed that the sensitivities of

258

using pCAN in the nine PCR assays could be as low as 15 genome copies per reaction

259

except for the assays of events RF2, MS1, and GT73, which have sensitivities of 25

260

copies per reaction (Figure 3). The sensitivity of 25 genome copies is approximately

261

equal to 0.05% GM DNA in 100 ng canola genomic DNA, which is accepted in the 14


Page 15 of 44


262

requirements of GMO labeling regulations around the world. 2, 30

263

Applicability of pCAN as calibrator in quantitative real-time PCR analysis

264

In order to evaluate the applicability of pCAN as calibrator in quantitative real-time

265

PCR assays of the eight GM canola events, primary real-time PCR parameters were

266

tested according to the guideline of real-time PCR method for GMOs, such as PCR

267

efficiency, linearity of standard curve, repeatability and reproducibility, limit of

268

detection (LOD), limit of quantification (LOQ), and conversion factor (Cf), etc. 30

269

PCR efficiencies and linearity of standard curves. Standard curves of real-time PCR

270

assays of the 10-fold serially diluted pCAN DNA solutions (2×105, 2×104, 2×103, 2×102,

271

and 20 copies/μL) were generated, and PCR efficiencies and linearity of the standard

272

curves were calculated. The PCR efficiencies of the eight event-specific GM canola

273

assays and endogenous PEP gene assay ranged from 91% to 97%, which were within

274

the range of 90–110 % recommended by the European Commission (Figure 4). 30 The

275

linearity of standard curves of the nine real-time PCR assays varied from 0.9987 to

276

1.0000 (Figure 4), indicating excellent linearity between the DNA copy numbers in

277

the template and corresponding fluorescent intensities (Ct). The high PCR efficiency

278

and good linearity of standard curves indicate that pCAN could be used as calibrator

279

in real-time PCR assays of the eight GM canola events and the endogenous PEP gene.

280

Repeatability and reproducibility. Repeatability and reproducibility of the nine

281

real-time PCR assays using pCAN as calibrator were evaluated by calculating the

282

relative standard deviation (RSD) of the nine repeats of one PCR assay (three times

283

and each time with three parallel replicates). To determine repeatability, the nine 15



Page 16 of 44

284

PCR reactions were performed by the same operator. For reproducibility, the nine

285

PCR reactions were performed by three different operators (each operator

286

performed PCR assay once with three parallel replicate reactions). As shown in Table

287

2, the standard deviation of repeatability (SDr) ranged from 0.01 to 0.41, and the

288

relative standard deviation of repeatability (RSDr) ranged from 0.05% to 1.60%. The

289

standard deviation of reproducibility (SDR) ranged from 0.04 to 0.39, and the relative

290

standard deviation of reproducibility (RSDR) ranged from 0.13% to 1.79%. The

291

acceptable RSDr and RSDR by ENGL guideline were ≤25%, 30 and our RSDr and RSDR

292

values are well below 25%, indicating pCAN has good repeatability and

293

reproducibility when employed as calibrator for the nine real-time PCR assays.

294

LOD and LOQ.

295

In real-time PCR analysis, the LOD and LOQ are the lowest genome copy number of

296

the target DNA that can be reliably detected and accurately quantified with a

297

confidence interval of more than 95%. To determine the LODs and LOQs of pCAN in

298

each assay, pCAN DNA solutions with concentrations of 4, 2 and 1 copy/μl) were

299

prepared as PCR templates, and 20 real-time PCR reactions for each concentration

300

was performed in parallel. For the nine real-time PCR assays (eight event-specific

301

assays and PEP assay), good PCR amplification curves and reliable Ct values were

302

observed in all 20 parallel reactions when 20 or 10 copies of pCAN DNA was used as

303

template, indicating that the LODs are as low as 10 genome copies for all 9 assays.

304

When 20 genome copies of pCAN DNA was used as template, the RSD values of the

305

20 parallel reactions ranged from 9.38% to 22.53%, and the quantification biases 16


Page 17 of 44


306

were between 1.34% and 14.36% (Table 3). However, when 10 genome copies of

307

pCAN DNA was used as template, the RSD values were higher than 25% (27.60% to

308

49.12%), even though the quantification biases for RF2, MS8, and Topas 19/2 assays

309

were lower than 25% (Table 3). Concluding from the abovementioned results, the

310

LOQs of the nine real-time PCR assays using pCAN plasmid DNA as templates are

311

determined to be as low as 20 genome copies.

312

Conversion Factor. The Cf value for pCAN in each event-specific GM canola assay was

313

determined using homogenous genomic DNAs of each event at three different

314

concentrations (10.0, 5.0, and 1.0 ng/μl). The results were listed in Table 4. Cf values

315

for RF1, RF2, MS1, MS8, Topas 19/2, Oxy235, RT73 and T45 event-specific

316

quantitative PCR assays were 4.63, 2.93, 0.87, 1.29, 1.31, 1.93, 2.04, and 2.04,

317

respectively; the RSD of these Cfs are ranged from 3.00% to 16.35%, far below 35%.

318

In theory, Cf values should be consistent with the value of 1.0 among different

319

event-specific assays if the endogenous reference gene is single copy in haploid

320

genome and there is no difference in PCR efficiency between using genomic DNA and

321

plasmid DNA as template. 14 Because of the complex genome of canola, the haploid

322

copy number of currently used endogenous reference genes, acetyl-CoA carboxylase

323

gene (BnACCg8), phosphoenolpyruvate carboxylase (PEP), oleoyl hydrolase gene

324

(FatA), high-mobility-group protein I/Y gene (HMG-I/Y) and cruciferin A gene (CruA),

325

were two copies at least, and the ideal Cf value should be 2.0. Our results showed

326

that the Cf values of MS8, Topas 19/2, Oxy235, RT73 and T45 were around 2.0, RF1,

327

RF2, and MS1 have slightly large variation, with the lowest Cf for MS1 at 0.87 and the 17



Page 18 of 44

328

highest cf value for RF1 at 4.63.

329

To assess the discrepancy between experimental and theoretical Cf values, we

330

constructed PCR standard curves (As shown in Figure 4 and Table S2) using genomic

331

DNA templates of each of the eight GM canola events at different concentrations

332

(50.0, 25.0, 5.0, 2.5, 0.5 ng/reaction). We found that the deviations of slopes and

333

intercepts of plasmid and genomic DNA standard curves (Table S2) might cause the

334

difference between the experimental and theoretical Cf values, especially for the

335

deviation of intercepts.31 For instance, in RF1 event and endogenous PEP gene assays,

336

standard curves between reactions using genomic DNA and pCAN plasmid DNA

337

templates had similar slopes (-3.4471 and -3.4466) but different intercepts of 39.583

338

and 37.052, the ⊿intercept of 2.531 was the mainly origin caused the high Cf value.

339

In RF1 event and endogenous PEP gene assays, the standard curves between

340

reactions using genomic DNA and pCAN plasmid DNA templates were almost

341

coincidnece resulted ideal Cf value. Also, one previous report on the comparison of

342

five canola endogenous reference genes indicated that the significant allelic variation

343

might cause the Ct values variation among different cultivars, such as HMG-I/Y and

344

BnACCg8. 32 The allelic variation of PEP gene among the eight GM canola events

345

might result differences of Cf values. Therefore, Cf values should be used to

346

normalize the ratio of exogenous to endogenous genome copies in quantitative

347

analysis of the eight GM canola events when using reference plasmid pCAN as

348

calibrator.

349

Performance of pCAN as a calibrator in quantitative analysis of canola samples 18


Page 19 of 44


350

To test the performance of pCAN as a reference calibrator in quantitative real-time

351

PCR analysis of GM canola, two sets of canola samples were tested. One set was

352

three simulated DNA samples (G1, G2 and G3) prepared by mixing GM canola

353

genomic DNA with non-GM canola genomic DNA at different ratios. The three

354

simulated samples are named G1, G2 and G3, which has 5.0%, 3.0%, and 1.0% GM

355

canola genomic DNA, respectively. The other set was ten canola seed samples

356

(named from R1 to R10) sampled from the shipped canola seeds to Shanghai port

357

and was kindly provided by Shanghai Entry-exit Inspection and Quarantine Bureau.

358

The accuracy and precision of the test results were used to evaluate the performance

359

of pCAN in real-time PCR assays of the canola samples. The accuracy of a test result

360

was measured as degree of bias, which was calculated using the formula of

361

|) -

|

-

× 100%. The precision of a test result was evaluated using the

362

standard deviation (SD) and relative standard deviation (RSD).

363

As shown in Table 5 and Figure 5, the mean of the tested values of the GM content in

364

each mixed canola genomic DNA sample using pCAN as the reference calibrator was

365

close to the actual value after applying corresponding Cf. The quantification bias of

366

all samples are ranged from 0.47% to 20.68%, which were less than the acceptance

367

criterion of

One Novel Multiple-Target Plasmid Reference Molecule Targeting

Recommend Documents