Development of certified matrix-based reference material as calibrator

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Development of certified matrix-based reference material as calibrator for genetically modified rice G6H1 analysis Yu Yang, Liang Li, Hui Yang, Xiaying Li, Xiujie Zhang, Junfeng Xu, Dabing Zhang, Wujun Jin, and Litao Yang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b00468 • Publication Date (Web): 27 Mar 2018 Downloaded from http://pubs.acs.org on March 28, 2018

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Journal of Agricultural and Food Chemistry

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Development of certified matrix-based reference material as

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calibrator for genetically modified rice G6H1 analysis

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Yu Yang1†, Liang Li2†, Hui Yang1, Xiaying Li3, Xiujie Zhang3, Junfeng Xu4, Dabing Zhang1,

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Wujun Jin2, Litao Yang1*

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1

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

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Shanghai 200240, China

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2

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National Center for the Molecular Characterization of Genetically Modified

Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing 100081, China

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3

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Republic of China. Beijing 100025, China.

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4

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Control, Institute of Quality and Standard for Agro-Products, Zhejiang Academy of

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Agricultural Sciences; Hangzhou 310021, China

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

State Key Laboratory Breeding Base for Zhejiang Sustainable Pest and Disease

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†These two authors contributed equally to this work.

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*

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34207174; Email: [email protected].

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

ACS Paragon Plus Environment


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Abstract

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The accurate monitoring and quantification of genetically modified organisms (GMOs)

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are key points for the implementation of labelling regulations, and a certified

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reference material (CRM) acts as the scaleplate for quantifying the GM contents of

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foods/feeds and evaluating a GMO analytical method or equipment. Herein, we

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developed a series of CRMs for transgenic rice event G6H1, which possesses

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insect-resistant and herbicide-tolerant traits. Three G6H1 CRMs were produced by

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mixing seed powders obtained from homozygous G6H1 and its recipient cultivar

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Xiushui 110 at mass ratios of 49.825%, 9.967%, and 4.986%. The between-bottle

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homogeneity and within-bottle homogeneity was thoroughly evaluated with

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consistent results. The potential DNA degradation in transportation and shelf life

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were evaluated with an expiration period of at least 12 months. The property values

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of three CRMs (G6H1a, G6H1b, G6H1c) were given as (49.825±0.448) g/kg,

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(9.967±1.757) g/kg, and (4.986±1.274) g/kg based on mass fraction ratio,

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respectively. Furthermore, the three CRMs were characterized with values of (5.01

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±0.08)%, （1.06±0.22)%, and (0.53±0.11)% based on the copy number ratio

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using droplet digital PCR method. All results confirmed that the produced G6H1

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matrix-based CRMs are of high quality with precise characterization values and can

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be used as calibrators in GM rice G6H1 inspection and monitoring and in evaluating

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new analytical methods or devices targeting G6H1 event.

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Keywords: Genetically Modified Organism, Certified Reference Materials, G6H1


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rice, Characterization value.



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Introduction

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With the development of transgenic technology, many more genetically modified

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(GM) crop events have been approved for planting around the world. By the end of

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2016, a total of 477 GM events of 29 plant species had been approved for

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commercialization, including maize, soybean, cotton, canola, potato, and rice. 1

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Although transgenic crops deliver substantial agronomic, environmental, and

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economic benefits to farmers and consumers, the public still has some concerns

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about the food safety of transgenic crops and their derivatives. Many countries and

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international organizations have issued guidelines and regulations to strengthen the

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commercialization and administration of GM foods and feeds, including procedures

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for risk assessment and labelling. 2 For example, the EU regulated that food/feed

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samples containing more than 0.9% GM contents should be labelled, but a zero

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threshold was set in China. 3-5 To effectively implement labelling regulations, the

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standardization of genetically modified organism (GMO) analysis is becoming

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increasingly necessary, including the harmonization of the plant endogenous

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reference genes, the validation of PCR and real-time PCR assays, and the

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development of certified reference materials (CRMs).6, 7

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A certified reference material (CRM) is a specific material that can be characterized

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by one or more properties specified by valid metrological procedures, and it has

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associated uncertainties that can be traced to the International System of Units. 8

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Because of their characteristics of accurate values and traceability, CRMs are mainly


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used as calibrators in the quantitative analysis of samples and in the performance

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evaluation of new methods or equipment in analytical fields such as chemistry,

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biology, medicine, and food safety. While the development of new molecular

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analytical techniques targeting proteins, nucleic acids, and metabolites has been

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rapid, the development of CRMs lags behind. 6 For example, there is a shortage of

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protein CRMs for allergen detection, RNA/DNA marker CRMs for disease diagnosis,

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and protein/DNA CRMs for the analysis of GM contents. 9-11 In GMO analysis, the

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Institute for Reference Materials and Measurements (IRMM) and the American Oil

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Chemists’ Society (AOCS) are currently the main CRMs developers. Genomic

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DNA-based CRMs, matrix-based CRMs, and plasmid DNA-based CRMs are three

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major types of GMO detection analysis. 12 Matrix-based CRMs are powder mixtures

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of GM powders and non-GM powders at the desired mass ratio; 6, 7, 13 the powders

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are made from original plant materials, such as seeds, stalks, and leaves. Genomic

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DNA-based CRMs are genomic DNA extracted from leaves of certified homozygous

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GM plants, and plasmid DNA-based CRMs are recombinant plasmids containing one

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or several exogenous DNA sequences and endogenous reference gene. 12, 14, 15

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Matrix-based CRMs are the most widely used type of CRM due to their similarities to

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blind samples and their easy traceability.6, 7 In addition, the determined value with

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the mass ratio is consistent with the required labelling thresholds in most countries.

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At present, more than 130 CRMs targeting 76 GM events have been developed and

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commercialized, including 19 genomic DNA-based CRMs, 101 matrix-based CRMs,

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and 13 plasmid DNA-based CRMs.1, 16 Most of these CRMs were developed for the



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analysis of GM maize, soybean, canola, and cotton events, but only a few exist for

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rice. Currently, eight GM rice events have been developed and authorized

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commercially in different countries: PWC16, CL121/CL141/CFX51 and

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IMINTA-1/IMINTA-4 have been approved in Canada; LLRICE06/LLRICE62 was

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approved in Australia, Canada, Colombia, Mexico, and the USA; LLRICE601 was

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commercialized in Colombia; Huahui No.1/Bt Shanyou 63 was commercialized in

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China; 17 Tarom molaii +Cry1Ab is used in Iran; and 7 Crp#10 was approved in Japan. 1,

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18, 19

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insect resistance, herbicide tolerance, disease resistance and nutrient improvement,

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and most of them are in the pipeline of safety production testing and

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commercialization. Among these commercialized GM rice events, only a genomic

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DNA CRM AOCS 0306-I for LLRICE62 and a matrix-based CRM for TT51-1 event have

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been developed. 20, 21 GM rice event G6H1 was developed by Zhejiang University and

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produced by integrating modified copies of the Cry1Ab/Vip3H and G6-EPSPS genes

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into rice cultivar Xiushui 110 using an Agrobacterium-mediated method. 22 The GM

In China, several new GM rice events have been produced with the traits of

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rice G6H1 was endowed with insect resistance and herbicide tolerance 22, 23 and is in

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the process of commercialization in China. Because G6H1 is likely to be planted in

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China, the analytical method and CRM for G6H1 identification should be developed

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at the same time. In this study, we developed a series of matrix-based CRMs with the

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desired GM contents for GM rice G6H1. The entire process, including the planting of

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candidate materials, candidate identification, seed harmonization, homogeneity

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assessment, stability assessment, and value characterization, is presented in this


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study.



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Materials and methods

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Plant materials and DNA extraction

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Homogenous GM rice G6H1 seeds and the seeds of its receptor (Xiushui 110 cultivar)

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were planted and harvested at the ZheJiang Academy of Agricultural Science (ZJAAS,

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China). The seeds of Xiushui 110 were confirmed without GM contents by ZJAAS,

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China. Seed samples of other GM rice events (G281, TT51-1, KMD, KF6, KF8, and M12)

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were supplied by their producers and were used as controls in the specificity testing

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of GM event G6H1. Conventional non-transgenic samples of maize, rice, canola and

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soybean were purchased from a local market in Shanghai, China, and were confirmed

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to be GMO-free by our lab. Samples of GM maize event NK603, GM soybean event

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MON89788, GM cotton event MON88913 and GM rapeseed event MS1 were

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provided by their developers. Plant genomic DNA samples were extracted and

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purified using the DNeasy Plant Mini Kit (Qiagen, Shanghai, China). The quality and

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quantity of the extracted DNA samples were evaluated by spectrometric assay using

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a Nanodrop 1000 (Thermo Scientific, Wilmington, DE, USA) and 1% agarose gel

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electrophoresis.

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Processing of GH61 matrix-based CRMs

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To avoid cross-contamination and/or foreign contamination, the G6H1 and non-GM

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Xiushui 110 base materials were processed independently in positive and negative

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preparation rooms with dedicated individual instruments before blending.


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Before grinding the seeds into powders, the base materials were pre-treated

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according to the following procedure: i) removing the impurities from the base

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materials, such as leaves, debris, dust, and sand; ii) washing the base materials with

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double-distilled water and retaining the plump-eared seeds; and iii) drying the

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retained seeds under a vacuum at 50 °C for 48 h. After pre-treatment, the seeds of

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GM rice G6H1 and non-GM Xiushui 110 were ground with a mill (6870 Freezer/Mill;

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SPEX SamplePrep, Metuchen, NJ, USA) with the following procedure: eight cycles of

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precooling for 2 min, milling for 3 min, and cooling for 2 min at a rate of 10 cycles per

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minute. During the grinding process, the milled samples were submerged in liquid

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nitrogen to maintain a low temperature. A standard sieve with a pore size of 800 μm

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was used to pre-sift the ground powder, and particles that did not pass through the

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sieve were further ground in the mill until the particle size was less than 800 μm. The

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ground seed powders were then vacuum-dried with a Labconco freeze dry system at

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-80 °C for 72 h to further decrease the water content. The final moisture content of

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the GM and non-GM rice seed powders was measured by thermogravimetric analysis

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with a Mettler Toledo HB43-s moisture meter. The particle size of the ground base

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materials was evaluated using a vibration sieve filter machine (8411-A, Hunan)

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equipped with a series of standard sieves with pore sizes ranging from 63 to 710 μm

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to ensure that the GM and non-GM base materials were of similar particle size.

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Particle size determination was repeated three times for both base materials.

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After determining the powder moisture content and particle size, the ground GM and

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non-GM base materials were mixed in desired ratios to produce three G6H1 CRMs of



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50.000 g/kg, 10.000 g/kg, or 5.000 g/kg. The GM and non-GM rice seed powders

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were weighed on a calibrated balance with a relative standard uncertainty less than

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0.1% (Supplementary Table S1). Weighed GM and non-GM powders were then

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mixed for 36 h at 25 rpm in an MR10L mixer (Chopin Technologies, France) to ensure

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homogeneity. Six 100-mg sub-samples were randomly sampled at different positions

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from the mixed materials and used to evaluate the initial homogeneity using

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real-time PCR analysis.

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An automatic filling device was used to transfer 1.00 g quantities of mixed powder

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into 10-mL amber glass vials. Before each vial was capped, air was evacuated using a

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freeze-drier and replaced by 99.9% pure argon. The vials were then sealed with

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aluminium caps. After inventory and selecting vials for further analysis in a random

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stratified sampling scheme, the vials were stored in a 4°C cold room.

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Property value and uncertainty evaluation

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Two types of property values were given to the G6H1 CRMs produced. One is the GM

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content based on mass fraction (GMm), and the other is the GM content based on

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the ratio of inserted exogenous versus endogenous genome copy number (GMc).

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GMm values and the corresponding uncertainty were calculated according to weight

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measurements, considering the moisture content, the purity of the base materials

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and measurement of weight. The GMc values were determined by absolute

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quantification using droplet digital PCR (ddPCR). The combined expanded

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uncertainties of the CRMs (UCRM) consist of uncertainties of characterization (Uchar),


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potential between-unit heterogeneity (Ubb), and potential degradation during

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long-term storage (Ults). The UCRM with coverage factor k was calculated by the

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following formula of U CRM

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Oligonucleotide PCR primers and probes

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All primer pairs (G6H1-F/G6H1-R, G6H1-F/Xiu-R) were designed according to the

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sequence flanking the exogenous DNA integration site and were used for

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homozygosity analysis of the G6H1 base materials. The primer pair G6H1-F/G6H1-R

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targeted the 5’ flanking sequence of GM rice G6H1, and the primer pair

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G6H1-F/Xiu-R targeted the 3’ flanking sequence. The primer pair of SPS-F/SPS-R with

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the combination of SPS-P probe and the primers qG6H1-F/qG6H1-R and qG6H1-P

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probe were designed and used to quantify the amounts of rice genome and G6H1

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event, respectively. All the primers and probes are listed in Supplementary Table S2

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and were synthesized by Invitrogen (Shanghai, China).

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Qualitative PCR

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Qualitative PCR assays employing G6H1-F/G6H1-R, G6H1-F/Xiu-R were performed to

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evaluate the homozygosity and purity of the base materials. The specificity of

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qualitative PCR assays of G6H1-F/G6H1-R and G6H1-F/Xiu-R were evaluated

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employing the genomic DNAs of different GM events (GM rice G281, TT51-1, KMD,

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KF6, KF8, and M12, GM maize event NK603, GM soybean event MON89788, GM

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cotton event MON88913, and GM rapeseed event MS1) and non-GM crops (rice,

2 2 = k × uchar + ubb2 + ults .



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maize, canola and soybean) as templates. The sensitivity of PCR assays

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(G6H1-F/G6H1-R and G6H1-F/Xiu-R) were tested using series of G6H1 and Xiushui

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110 genomic DNA dilutions with the corresponding contents of 5.0%, 1.0%, 0.5%,

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0.1%, and 0.05%, respectively. PCRs were carried out using a Veriti Thermal Cycler

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(Applied Biosystems, Carlsbad, CA, USA) in a 30 μL volume. Each reaction was

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composed of 2 ng genomic DNA template, 50 μm dNTPs, 250 nm primers, 0.5 U Taq

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DNA polymerase (Takara Bio, China) and 1X PCR buffer. The PCRs were run with the

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following program: denaturation at 94°C for 5 min; 35 cycles at 94°C for 30 s, 30 s at

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58°C, and 30 s at 72°C; and a final extension step for 5 min at 72°C. The PCR products

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were analysed on a 2% agarose gel after electrophoresis for approximately 30 min at

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150 V using GelRed stain for visualization.

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Real-time PCR

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Real-time PCR assays of G6H1 event rice and the SPS endogenous gene were

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performed to evaluate the homogeneity, stability, and minimum sampling amount of

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the G6H1 CRMs. The genomic DNA samples extracted from homogenous G6H1 rice

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leaves were diluted to different concentrations (100.0, 10.0, 1.0, 0.1 and 0.01 ng/uL)

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and used as calibrators for constructing the standard curve and evaluating the

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sensitivity in G6H1 event real-time PCR analysis. The specificity of G6H1 event

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real-time PCR assay was tested employing several GM events, such as GM rice events

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(G281, TT51-1, KMD, KF6, KF8, and M12), GM maize event NK603, GM soybean event

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MON89788, GM cotton event MON88913, and GM rapeseed event MS1. Real-time


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PCR reactions were performed in a 25 μL volume. The reactions contained 1 X PCR

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buffer, 200 μM dNTPs, 400 nM primer mixture, 200 nM TaqMan probe, 1.25 U Taq

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DNA polymerase, and DNA templates. Real-time PCRs were run on a fluorescent

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thermal cycler (ABI7900, Applied Biosystems, Carlsbad, CA, USA) with the following

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program: 95°C for 10 min, followed by 45 cycles of 15 s at 94°C and 60 s at 60°C.

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Fluorescent signal was monitored during the annealing step of each cycle. Data were

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analysed using the ABI SDS 2.0 Detection System. Each reaction was performed with

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three replicates, and each PCR analysis was repeated three times.

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Droplet digital PCR

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Droplet digital PCR (ddPCR) analysis was used for the absolute quantification of the

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GM content of the produced G6H1 CRMs. Each reaction was carried out in a volume

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of 20 μl containing 1 μl of forward/reverse primers (final concentration: 10 μM), 0.5

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μl of TaqMan probe (final concentration 10 μM), 10 μl of ddPCR Mix (2X), and 1 μl of

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diluted genomic DNA. ddPCR was performed on QX200 Droplet Digital PCR (ddPCR™)

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System (Bio-Rad, Inc. USA) as follows: 94°C for 5 min, followed by 45 cycles of 94°C

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for 15 s and 60°C for 1 min. The amplified droplets were then analysed using

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QuantaSoft. Each reaction was repeated three times with three replicates each time.



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Results

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Homozygosity and purity of the G6H1 and Xiushui 110 base materials

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GM event G6H1 and its recipient cultivar Xiushui 110 were planted in completely

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isolated fields for two generations by ZJAAS, China. Based on the sequences flanking

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the exogenous insert, we designed three PCR primers (G6H1-F, G6H1-R, and Xiu-R)

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specific to the 5’ and 3’ flanking regions to identify and confirm the purity and

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homozygosity of the harvested materials (as shown in Figure 1). For homozygous

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G6H1 leaf or seed samples, a 188 bp DNA fragment can be amplified using the

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G6H1-F/G6H1-R primer pair. In non-GM Xiushui 110, a 357 bp DNA fragment can only

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be amplified using the G6H1-F/Xiu-R primer pair. In heterozygous G6H1 leaf or seed

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samples, both the 188 bp and 357 bp fragments can be amplified using the primers

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G6H1-F/G6H1-R and G6H1-F/Xiu-R.

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The high specificity of designed primer pairs of G6H1-F/G6H1-R and G6H1-F/Xiu-R

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were well evaluated employing different GM events and non-GM crops. The results

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showed that only one 188 bp amplicon was observed in the PCR reaction of

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G6H1-F/G6H1-R using G6H1 genome DNA as template, and no DNA amplicons were

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observed in recitations with the DNA template of GM rice G281, TT51-1, KMD, KF6,

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KF8, and M12, GM maize event NK603, GM soybean event MON89788, GM cotton

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event MON88913, and GM rapeseed event MS1. In the PCR reaction using

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G6H1-F/Xiu-R, the 357 bp DNA fragments were only obtained from rice samples, and

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no amplicon in the reactions of maize, canola and soybean (Supplementary Figure


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S1a and 1b). Also, the relative limit of detection (LOD) of the qualitative PCRs with

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G6H1-F/G6H1-R and G6H1-F/Xiu-R were tested with the values of 0.1% in mass ratio

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(Supplementary Figure S1c and 1d).

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To obtain base materials of high homozygosity and purity, leaves were sampled from

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each G6H1 and Xiushui 110 plant and tested at each generation. Only homozygous

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G6H1 plants and non-GM Xiushui 110 plants were retained for harvest. The G6H1

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seeds and Xiushui110 seeds were obtained through three generations of planting

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and verification, respectively. In each generation, the ears of each plant were bagged

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and self-crossed to harvest seeds for the next generation. 800 plants of G6H1 event

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and 1000 plants of Xiushui 110 were kept for harvest in the first generation,

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respectively. In the second generation, 1500 G6H1 plants and 3000 Xiushui 110

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plants were kept for harvest. After three generation, 50 Kg of homozygous seeds

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were harvested. The final harvested G6H1 and Xiushui110 seeds were used as base

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materials. To further determine the purity and homozygosity of the harvest base

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materials, 3000 seeds were randomly sampled and tested from G6H1 and Xiushui

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110 plants. To decrease the cost and time of testing, we designed a group testing

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strategy to assay the sampled seeds. The 3000 seeds were first divided into 30

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groups (100 seeds per group), and each group was tested as one batch sample. Next,

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the groups producing unexpected results were selected for testing of individual

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seeds. The 30 groups were coded G1 to G30 for G6H1 and X1 to X30 for Xiushui 110.

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For all 30 G6H1 groups, the expected 188 bp fragment was amplified using the

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G6H1-F/G6H1-R primers, and no expected fragment was obtained using the



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G6H1-F/Xiu-R primers (Supplementary Figure S2a and 2b), indicating that all G6H1

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seeds in each group were homozygous. Because the limit of detection of

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conventional PCR is tested with the value of 0.1%, these results suggested that all

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G6H1 seeds were homogenous, with a purity of greater than 99.9% at the 95%

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confidence level. In PCR tests of the 30 groups of Xiushui 110 seeds, the expected

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357 bp fragment was amplified with the G6H1-F/Xiu-R primers, and no 188 bp

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fragment was observed using the G6H1-F/G6H1-R primers (Supplementary Figure

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S2c and 2d). This result indicated that there was no G6H1 seed contamination and

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that the purity of Xiushui 110 seeds was higher than 99.9% at the 95% confidence

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level.

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Processing G6H1 CRMs with expired GM contents

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Due to the great effect of particle size and moisture content on the homogeneity,

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stability and extraction efficiency of genomic DNA, GM and non-GM base materials

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must be ground into small and symmetrical powders of similar size distribution. The

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particle size of ground GM and non-GM powders was measured to evaluate the

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particle size distribution. The results showed that more than 70.0% particles were

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smaller than 355 μm and that approximately 60% were smaller than 250 μm (as

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shown in Supplementary Figure S3). According to ANOVA analysis, there was no

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significant difference in particle size distribution between the powders. These results

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suggest that the G6H1 and Xiushui 110 ground powders are suitable for further

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blending and producing matrix-based CRMs.


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The moisture values of the two powders were evaluated. Five subsamples of 2 g each

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were used to test water content. The mean moisture values of the G6H1 powders

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and Xiushui 110 powders were 2.59% and 2.21%, respectively (Supplementary Table

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S3). The moisture values of G6H1 and Xiushui 110 were very close and met the

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requirements of CRMs producing.

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After the particle size distributions and moisture values were confirmed, we

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produced three G6H1 CRMs (G6H1a, G6H1b, and G6H1c) with the desired GM

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concentrations by blending and thoroughly mixing G6H1 and Xiushui 110 powders

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(Supplementary Table S1). A total of 500 vials was packaged for each CRM.

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Minimum Sample Intake Analysis

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The minimum sample intake is closely related to the homogeneity of a CRM,

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especially for CRMs with lower GM contents. To determine the minimum sample

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intake of the G6H1 CRMs, a G6H1c CRM with an expired content of 0.5% was used. A

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total of 36 vials were sampled from bottled G6H1c CRMs. These 36 vials were then

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divided into 4 groups (9 vials/group). Powders were sampled at 20 mg, 50 mg, 100

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mg, and 200 mg from each vial in each corresponding group for DNA extraction and

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G6H1 content quantification. All extracted genomic DNA samples were diluted to a

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final concentration of 10 ng/µL for use as templates in G6H1 event-specific real-time

311

PCR assays. The high specificity and sensitivity of the G6H1 event real-time PCR assay

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were well evaluated (Supplementary Figure S4). The quantified results showed that

313

slightly large bias values of 6.15 and 15.63 were obtained in samples with intakes of



314

20 mg and 50 mg, respectively. Ideal GM contents of approximately 0.5% (range: 0.48

315

to 0.55) were obtained in samples with intakes of 100 mg and 200 mg, and the bias

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values were as low as 0.02 (Figure 2). The F-test of the quantified results from 4

317

groups showed that there were no differences in the intakes of the 100 mg and 200

318

mg samples (Supplementary Table S4). Based on the quantified results, we

319

determined that the minimum sample intake should be 100 mg in practical

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application.

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Homogeneity Analysis

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To evaluate the homogeneity of the G6H1 CRMs, 15 vials were randomly selected for

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between-bottle homogeneity analysis. Three subsamples of 100 mg each were

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collected for the analysis of within-bottle homogeneity according to ISO Guide

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35:2006. 24 Within-bottle homogeneity is a measure of the potential minimum

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sample intake, and between-bottle homogeneity indicates bottle-to-bottle

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variation.25 For each sample, genomic DNA was extracted and diluted to a final

328

concentration of 10 ng/μL for further analysis. The GM content of each sample was

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calculated based on the Ct values calculated based on G6H1 and SPS real-time PCR

330

assays.26, 27 Homogeneity was evaluated using the quantitative data via the F-test. All

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real-time PCR data are shown in Figure 3, and the relative standard deviation (RSD)

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was evaluated with respect to an acceptable critical value of 25%.28 F-test values for

333

all three CRMs were less than the critical F0.05 (14,30) value of 2.04 (Table 1). These

334

statistical analysis results suggest that a homogeneous batch was produced for each


Page 18 of 37

Page 19 of 37


335

CRM and that the standard deviation of homogeneity was far less than the standard

336

deviation of real-time PCR. In addition, the uncertainty resulting from potential

337

undetected heterogeneity (ubb) was calculated. The ubb values of the G6H1a, G6H1b,

338

and G6H1c CRMs were 0.010%, 0.017%, and 0.015%, respectively.

339

Stability study

340

Stability is an important index of CRM quality and affects the conditions of

341

short-term and long-term storage. Stability was evaluated using an isochronous

342

design consisting of simultaneous analysis of samples from both reference and test

343

vials.29 In the short-term stability test, the possible external influences on the

344

stability of the CRMs at different temperatures during transportation were evaluated.

345

The long-term stability test was performed to determine the stability of the samples

346

after extended storage under optimum storage conditions.25 In this study, five vials of

347

CRMs were used at each temperature/time combination for both short- and

348

long-term stability tests. At the end of each test, all five vials were quantified by

349

real-time PCR assays in triplicate, and the data were analysed and used to draw a

350

regression line for GM content as a function of time.

351

In the short-term stability test, the vials were stored at -20°C, 4°C, or 26°C, for 0, 1, 2,

352

or 4 weeks. The quantified GM content of each vial is shown in Supplementary

353

Figure S5. No outlier values were detected by the Grubbs test. Regression analysis

354

was performed for each of the storage temperatures to reveal any trend in GM

355

content in relation to the duration of storage. The results of the t-test showed that



356

there was no significant slope for the vials at the three temperatures at the 95%

357

confidence level (Supplementary Table S5), suggesting that the uncertainty in the

358

certified values after 4 weeks can be ignored compared to the overall uncertainty.

359

For the long-term stability test, the vials were preserved for 0, 1, 2, 4, 6 or 12 months

360

at 4°C or -20°C. Five vials were tested in triplicate at each temperature/time

361

combination. The quantified GM content results are shown in Figure 4. The t-test

362

data illustrate that there are no obvious effects on the regression slope at either

363

temperature at the 95% confidence level (Table 2). The relative uncertainty in

364

stability for each CRM at -20 °C was calculated, resulting in values of 0.019%, 0.081%

365

and 0.019%, respectively. Based on these results, we believe that the produced G6H1

366

CRMs can be stored at 4°C or -20°C for at least 12 months.

367

Property value determination

368

The G6H1 CRMs were characterized with two type values, considering the GMO

369

labelling regulations and practical application. One was presented by the mass

370

fraction ratio (GMm), and the other was the copy number ratio (GMc).

371

The GMm values were calculated using the formula of

372

GMm=(௠

373

where mG6H1 and mXiushui are the amounts of weighed G6H1 and Xiushui powders,

374

⊿mG6H1 and ⊿mXiushui are the moisture contents of the G6H1 and Xiushui powders,

375

and ρG6H1 and ρXiushui are the purities of the G6H1 and Xiushui 110 seeds. The purities

(௠ಸలಹభ ି∆௠ಸలಹభ )∗ఘಸలಹభ ಸలಹభ ି∆௠ಸలಹభ )∗ఘಸలಹభ ା(௠೉೔ೠೞ೓ೠ೔ ି∆௠೉೔ೠೞ೓ೠ೔ )∗ఘ೉೔ೠೞ೓ೠ೔


Page 20 of 37

Page 21 of 37


376

of the G6H1 and Xiushui 110 seeds were certified as 99.9% using qualiative PCR at

377

the 95% confidence level. The GMm values of the three G6H1 CRMs were 49.825

378

g/kg, 9.967 g/kg, and 4.986 g/kg, respectively. The uncertainty caused by weight and

379

moisture measurements (uchar) was also evaluated (Table 3).

380

To characterize CRMs based on genome copy ratio, droplet digital PCR assays of the

381

G6H1 rice event were established and used for absolute quantification. For each

382

G6H1 CRM, five vials were randomly sampled for GM content quantification, and

383

each vial was tested in triplicate. The 15 independent values were then examined for

384

each CRM, and the mean value was designated the certified value (Figure 5). The

385

certified values obtained for G6H1a, G6H1b, and G6H1c were 5.01%, 1.06%, and

386

0.53%, respectively (Table 4). The uncertainty (uchar) of the characterization was

387

calculated using the formula u char =

388

G6H1b, and G6H1c were 0.032%, 0.069%, and 0.047%, respectively (Table 4).

t 0.05 (n − 1) × s n

. The uchar values of G6H1a,

389



Page 22 of 37

390

Discussion

391

GM CRMs differ from conventional chemical CRMs in that the quantification of GM

392

contents is performed by indirect measurement of DNA, whereas the actual

393

quantification is based on the genome copy number ratio and mass fraction ratio. 21

394

The GM and non-GM materials used in producing matrix-based CRMs must be

395

prepared under very strict guidelines21, including the high purity and zygosity of both

396

the GM and its recipient non-GM material. In this study, strategies for genotyping,

397

GM event detection, and homozygosity determination were established and used to

398

prepare the base materials of G6H1 and its recipient line Xiushui 110. In previously

399

reported CRMs, heterozygous GM seeds were often used and assigned a property

400

value based on the mass fraction ratio. However, these CRMs might result in the

401

inaccurate quantification of practical samples when used as calibrators according to

402

the DNA quantification method. 30 For heterozygous GM rice seeds, the genome copy

403

number ratio between exogenous and host endogenous DNA will differ depending

404

on whether the GM event originated from the male or female parent. Liu et al.

405

reported that the GM content (genome copy number ratio) of heterozygous

406

transgenic rice seeds was not exactly half of the homozygous seed and that the

407

detailed GM content of the seed depended on the origin of the transgenic parent.

408

Therefore, we selected homozygous G6H1 seeds for producing matrix-based CRMs to

409

avoid GM content variation between the mass fraction ratio and genome copy

410

number ratio.


30

Page 23 of 37


411

For most previously developed matrix-based CRMs, the property value is presented

412

as a mass fraction ratio; few GM CRMs have been certified based on the genome

413

copy number ratio. The CRM mass fraction ratio is determined by accurately

414

weighing the candidate materials, whereas the genome copy number ratio is often

415

determined by real-time PCR in a multi-lab collaborative ring trial.6, 7 To determine

416

genome copy number ratio values by real-time PCR methods, calibrators with known

417

characterization values are required, and the accuracy and precision of the certified

418

value are dependent on the calibrators used. To certify the accurate property values

419

of copy number ratio for G6H1 CRMs, droplet digital PCR (ddPCR) with the function

420

of absolute quantification and without additional calibrators was used in this work. In

421

ddPCR analysis, the standard deviation (SD) and relative standard deviation (RSD)

422

values were smaller than those of real-time PCR methods, indicating that ddPCR has

423

better repeatability and reproducibility.26, 27 With the development of new platforms

424

with better precision and accuracy, digital PCR techniques have become the primary

425

method by which GM CRMs are certified.

426

In this study, three matrix-based G6H1 CRMs were produced and characterized by

427

both mass fraction ratio (GMm) and genome copy ratio (GMc). The certified GMm

428

values of G6H1a, G6H1b, G6H1c are 49.825 g/kg, 9.967 g/kg, and 4.986 g/kg,

429

respectively. The GMc values of G6H1a, G6H1b, and G6H1c are 5.01%, 1.06%, and

430

0.53%, respectively. The bias between GMm and GMc values is minor; thus, in

431

practical application, accurate quantification can be obtained using these G6H1 CRMs

432

no matter which characterization value (mass fraction ratio or genome copy number



433

ratio) is used.

434

G6H1 rice is insect-resistant and herbicide-tolerant, and it has great potential to

435

obtain a certificate of safe production for further commercialization. The

436

development of CRMs for G6H1 will help reduce the potential risk of contamination

437

during its planting and production and will aid in its inspection and regulation.

438 439

Acknowledgments

440

This work was supported by the National Transgenic Plant Special Fund

441

(2016ZX08012-003), the National Natural Science Foundation of China (31471670),

442

and Program for New Century Excellent Talents in University.

443


Page 24 of 37

Page 25 of 37


444

Figures

445

Figure 1. The homozygosity identification scheme used for GM rice G6H1 and its

446

recipient Xiushui 110. The locations of the primers used are provided.

447

Figure 2. Minimum sample intake analysis of G6H1c matrix-reference materials

448

Figure 3. The quantified G6H1 contents of sampled CRMs from quantitative real-time

449

PCR assays in homogeneity assessment. For each level, five vials were tested, and

450

three replicates were performed for each vial.

451

Figure 4. The quantified G6H1 contents of sampled CRMs from by quantitative

452

real-time PCR in stability assessment.

453

Figure 5. The absolutely quantified GM contents of G6H1 CRMs by digital PCR assay.

454

Five vials were randomly sampled, and each vial was tested in triplicate.

455



456

Figure 1

457 458

459

460

Figure 2

461 462

463

464


Page 26 of 37

Page 27 of 37


465

Figure 3

466 467

468

Figure 4

469 470

471

472



473

Figure 5

474


Page 28 of 37

Page 29 of 37


475

Tables

476

Table 1.Homogeneity assessment of G6H1 CRMs using the F test Name

G6H1a

Origin

SSD

df

MS

Within vial

0.151

14

0.104

Between

1.010

30

0.183

Within vial

0.045

14

0.056

Between

0.067

30

0.047

Within vial

0.024

14

0.042

Between

0.047

30

0.040

F

ubb (100%)

0.32

0.010

1.43

0.017

1.10

0.015

vials

G6H1b

vials

G6H1c

vials

477

Table 2. Statistical analysis of a long-term stability assessment of quantified

478

genetically modified content -20℃

4℃

Name

479

b1

S(b1)

b1

S(b1)

G6H1a

-0.0058

0.0080

-0.0087

0.0051

G6H1b

-0.0054

0.0071

-0.0029

0.0040

G6H1c

-0.0026

0.0008

-0.0003

0.0010

b1: slope of the regression line; S(b1): standard deviation of b1



480

Page 30 of 37

Table 3. Certified characterization values of G6H1 CRMs based on mass ratio Name

GMm (g/Kg)

uchar(g/Kg)

ubb(g/Kg)

ults(g/Kg)

uCRM,(g/Kg)k=2

G6H1a

49.825

0.050

0.100

0.190

0.448

G6H1b

9.967

0.290

0.170

0.810

1.757

G6H1c

4.986

0.590

0.150

0.190

1.274

481

482

Table 4. Certified characterization values of G6H1 CRMs based on copy number

483

ratio Name

GMc (%)

uchar

ubb

ults

uCRM,k=2

G6H1a

5.01

0.032

0.010

0.019

0.08

G6H1b

1.06

0.069

0.017

0.081

0.22

G6H1c

0.53

0.047

0.015

0.019

0.11

484


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485

486 487

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9. Cryar, A.; Pritchard, C.; Burkitt, W.; Walker, M.; O'Connor, G.; Burns, D. T.

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wine as a model system for metrologically traceable mass spectrometric

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10. Branford, S.; Fletcher, L.; Cross, N.; Müller, M.; Hochhaus, A.; Kim, D. Desirable

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11. Trapmann, S.; Emons, H. Reliable GMO Analysis. Anal. Bioanal. Chem. 2005, 381, 72-74.

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12. Pi, L.; Li, X.; Cao, Y.; Wang, C.; Pan, L.; Yang, L. Development and application of

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a multi-targeting reference plasmid as calibrator for analysis of five genetically

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modified soybean events. Anal. Bioanal. Chem. 2015, 407, 2877-2886.

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13. Broothaerts, W.; Corbisier, P.; Emons, H.;Emteborg, H.; Linsinger, T.; Trapmann,

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S. Development of a certified reference material for genetically modified

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potato with altered starch composition. J. Agric. Food Chem. 2007, 55,

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4728-4734.


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14. Zhang, H.; Yang, L.; Guo, J.; Li, X.; Jiang, L.; Zhang, D. Development of one

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novel multiple-target plasmid for duplex quantitative PCR analysis of roundup

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ready soybean. J. Agric. Food Chem. 2008, 56, 5514-5520.

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15. Yang, L.; Guo, J.; Pan, A.; Zhang, H.; Zhang, K.; Wang, Z. Event-specific

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quantitative detection of nine genetically modified maizes using one novel

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standard reference molecule. J. Agric. Food Chem. 2007, 55, 15-24.

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16. Wei, D.; Yang, L.; Shen, K.; Banghyun, K.; Kleter, G.; Marvin Hans, J. GMDD: a database of GMO detection methods. BMC Bioinf. 2008, 9, 260.

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17. Lu, C. The first approved transgenic rice in china. GM Crops. 2010, 1, 113-115.

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18. ISAAA. http://www.isaaa.org/gmapprovaldatabase/crop/default.asp?CropID=

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17&Crop=Rice

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19. GM Crop Database. http://www.cera-gmc.org/GMCropDatabase

534

20. Certificate of Analysis AOCS 0306-15, LLRice62 rice leaf tissue genomic DNA.

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http://aocs.files.cms-plus.com/TechnicalPDF/CRMs/030615_C.pdf.

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21. Jiang, Y.; Yang, H.; Quan, S.; Liu, Y.; Shen, P.; Yang, L. Development of certified

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matrix-based reference material of genetically modified rice event TT51-1 for

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real-time PCR quantification. Anal. Bioanal. Chem. 2015, 407, 6731-6739.



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22. Tian, J.; Liu, Z.; Chen, M.; Chen, Y.; Chen, X.; Peng, Y. Laboratory and field

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assessments of prey-mediated effects of transgenic BT rice on ummeliata

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insecticeps (Araneida: linyphiidae). Environ. Entomol. 2010, 39, 1369-1377.

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23.Lu, Z.; Han, N.; Tian, J.; Peng, Y.; Cui, H.; Guo, Y. Transgenic

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cry1Ab/vip3H+epsps rice with insect and herbicide resistance acted no

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adverse impacts on the population growth of a non-target herbivore, the

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white-backed planthopper, under laboratory and field conditions. J. Integr.

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Agric. 2014, 13, 2678-2689.

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24. International Organization of Standardization (2007) ISO Guide35:2006 – Reference materials—general and statistical principles for certification.

549

25. International Organization for Standardization (2008) ISO Guide 30:1992/Amd

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1:2008 – Revision of definitions for reference material and certified reference

551

material.

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26. Wu, Y.; Yang, L.; Cao, Y.; Song, G.; Shen, P.; Zhang, D. Collaborative validation

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of an event-specific quantitative real-time PCR method for genetically

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modified rice event TT51-1 detection. J. Agric. Food Chem. 2013, 61,

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5953-5960.

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27. Jiang, L.; Yang, L.; Zhang, H.; Guo, J.; Mazzara, M.; Eede, G. International collaborative study of the endogenous reference gene, sucrose phosphate


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synthase (SPS), used for qualitative and quantitative analysis of genetically

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modified rice. J. Agric. Food Chem. 2009, 57, 3525-3532.

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28. Marchesi, U.; Mazzara, M.; Broll, H.; Giacomo, M.; Grohmann, L.; Herau, V.

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European network of GMO laboratories (ENGL) Definition of minimum

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performance requirements for analytical methods of GMO testing. 2015.

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29. Lamberty, A.; Schimmel, H.; Pauwels, J. The study of the stability of reference

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materials by isochronous measurements. Fresen. J. Anal. Chem. 1998, 360,

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359-361.

566

30. Liu, D.; Shen, J.; Yang, L.; Zhang, D. Evaluation of the impacts of different

567

nuclear DNA content in the hull, endosperm, and embryo of rice seeds on GM

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rice quantification. J. Agric. Food Chem. 2010, 58, 4582-4587.



569

Supporting Information

570

Weight ratio results of G6H1 CRMs (Table S1); Primers and probes used for G6H1

571

CRMs development in qualitative PCR, real-time P, ddPCR analysis (Table S2); Water

572

content of GM rice G6H1 and non-GM Xiushui110 seed powders (Table S3);

573

Minimum sample intake analysis of G6H1c matrix reference material (Table S4); The

574

statistical analysis of the quantified genetically modified contents in short-term

575

stability assessment (Table S5); Specificity and sensitivity test of qualitative PCR

576

assays of G6H1-F/G6H1-R and G6H1-F/Xiu-R (Figure S1); Homozygosity and purity

577

test of the G6H1 and Xiushui 110 base materials (Figure S2); Histogram of particle

578

size distribution of ground G6H1 and Xiushui 110 seed powders (Figure S3); The

579

specificity and sensitivity test of G6H1 event real-time PCR assay (Figure S4); The

580

quantified GM contents of G6H1 CRM in quantitative real-time PCR for a 4-week

581

stability study (Figure S5).

582


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TOC

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Development of certified matrix-based reference material as calibrator

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