Development and Inter-laboratories Validation of Event-Specific

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New Analytical Methods

Development and Inter-laboratories Validation of Event-Specific Quantitative Real-Time PCR Method for Genetically Modified Rice G6H1 Event Litao Yang, Yu Yang, Wujun Jin, Xiujie Zhang, Xiaying Li, Yuhua Wu, Jun Li, and Liang Li J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01519 • Publication Date (Web): 09 Jul 2018 Downloaded from http://pubs.acs.org on July 9, 2018

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Journal of Agricultural and Food Chemistry

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Development and Inter-laboratories Validation of Event-Specific

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Quantitative Real-Time PCR Method for Genetically Modified Rice

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G6H1 Event

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Litao Yang1*, Yu Yang1, Wujun Jin2, Xiujie Zhang3, Xiaying Li3, Yuhua Wu4, Jun Li4,

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Liang Li2*

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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

National Center for the Molecular Characterization of Genetically Modified

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100081, China

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Republic of China. Beijing 100025, China.

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4

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Institute, Chinese Academy of Agricultural Sciences, No. 2 Xudong 2nd Road, Wuhan

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430062, People’s Republic of China

Biotechnology Research Institute, Chinese Academy of Agricultural Sciences, Beijing

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

Key Laboratory of Oil Crop Biology of the Ministry of Agriculture, Oil Crops Research

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*

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[email protected] (Liang Li).

To whom correspondence should be addressed: [email protected] (Litao Yang) or

ACS Paragon Plus Environment


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Abstract

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The transgenic rice G6H1 was a new event with the traits of herbicide-tolerance and

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insect-resistant. Herein, we developed one event-specific real-time PCR method with

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high specificity and sensitivity for G6H1 event quantitative analysis, and validated its

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performance on practical samples quantification through a collaborative ring trial. A

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total of eight laboratories participated in this validation and quantified three blind

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G6H1 powder samples including DNA extraction and real-time PCR analysis. The

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statistical analyzed results from returned data confirmed its high PCR efficiency, and

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good linearity, trueness and precision, indicating that the developed G6H1 real-time

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PCR assay were accurate, reliable, and comparable for G6H1 identification and

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quantification.

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Keywords: Collaborative ring trial; transgenic rice G6H1; Real-time PCR


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Introduction

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Rice is a staple food enriched with protein, vitamins and minerals, and which is

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widely planted in the world, especially in Southeast Asia and East Asia. However,

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some problems about rice planting that exist and disturb long are infestation of

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vermin and the use of herbicide, which bring huge damage to rice harvest and

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environment pollution.1 In recent years, DNA recombinant technique is widely

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applied in agriculture and enables continuous increase of genetically modified

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organisms (GMOs). Update to the end of 2016, 185.1 million hectares of GM crops

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have been planted in global area, more than 450 genetically modified (GM) events

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from 29 crop species have been developed and approved for commercialization.2 In

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GMOs, exogenous genes are integrated into recipient genome, and which can create

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one or more traits, such as insect-resistant and herbicide-tolerant.3 With great

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progress in rice genetic modification, more and more GM rice events with different

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traits have been developed.1, 4-5 Eight GM rice events have been approved for

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commercialization in several countries, such as PWC16, CL121/CL141/CFX51 and

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IMINTA-1/IMINTA-4 in Canada, LLRICE06/LLRICE62 in Australia, Canada, Colombia,

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Mexico, and the USA. LLRICE601 in Colombia, and Huahui No.1/Bt Shanyou 63 in

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China, Tarom molaii +Cry1Ab in Iran and 7 crp#10 in Japan.2, 6-7 In China, the

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transgenic rice research with the purpose of high insect-resistance,

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herbicide-tolerance, yields and quality is one hot topic because rice is one primary

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alimentary crop.1 Several transgenic rice events with the trait of insect-resistant have

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been developed in China, such as TT51-1, Kemingdao, KF6, T1C-19, and T2A-1, etc. 1, 5

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GM rice G6H1 developed by Zhejiang University is a novel event, which was

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produced by inserting a modified Cry1Ab/Vip3H gene encoding insect-resistant HJC-1



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protein and a G6-EPSPS gene encoding herbicide-tolerant protein. 8-10 The G6H1 rice

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showed very high insect-resistance and herbicide tolerance in the phases of field test

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and environment, and which are in the pipeline of applying the safety production

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certificate.8

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However, with the large-scale applications of transgenic technology in modern

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agricultural production, several controversial issues are being discussed including

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food safety, environment risk and ethical concerns.3-4 Several countries and areas

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have issued labeling regulations to protect the authority of consumers.11 For the

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implement of GMO labeling regulations, rapid and accurate detection methodologies

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of GM content have been developed.11 DNA-based PCR techniques were mainly used

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in GMO identification and quantification, such as PCR,12 real-time PCR,13-16 and digital

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PCR, etc.17-19 Real-time PCR employing the TaqMan probe technique is the golden

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standard for GMO analysis because of high sensitivity, accuracy, and practicability. A

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lot of real-time PCR assays were developed and validated to detect and quantify GM

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contents in raw materials and processed food/feed samples.20-25 Also, the

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collaborative ring trial validation is one prerequisite step for proposing a method to

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be used in routine lab analysis and included as one national or ISO standard.26-27

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According to the requests of GMO regulations in China and EU, the validated

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detection method for one GM event should be supplied before its application for

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commercialization.11 In this study, one event-specific real-time PCR method for G6H1

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rice inspection and monitoring were developed based on the 3’ junction sequence

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between exogenous DNA and rice DNA, and well validated employing three blind

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samples by a collaborative ring trial.


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Material and Methods

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Materials and DNA extraction

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Homozygous seeds of G6H1 rice and its recipient material Xiushui110 were grown

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and supplied by Zhejiang Academy of Agricultural Science (ZJAAS). The seed samples

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of GM rice TT51-1, T1C-19, Kemingdao, and KF6 were supplied by their developers.

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The mixed seed powder samples of GM maize (MON810, MON863, and Bt176), GM

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cotton (MON531 and MON88913), GM soybean (GTS40-3-2 and MON89788), and

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GM canola (Ms1Rsf1, Ms3Rf83, and GT73) were prepared by our laboratory. Three

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G6H1 matrix-based certified reference material (CRM) samples with the GM

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mass/mass ratio of 4.983%, 0.997%, and 0.499% were produced by Shanghai Jiao

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Tong University (SJTU), China and used as blind samples in collaborative ring trial.28

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All plant genomic DNAs were extracted firstly by CTAB extraction according to the

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standard of ISO 21571:2005 and purified using DNeasy 96 Plant Kit (Qiagen, Hilden,

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Germany).29 The quality and concentration of purified DNAs were measured and

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estimated using 1.0% agarose gel electrophoresis and the ultraviolet spectrometric

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method with a NanoDrop 1000 UV/vis spectrophotometer (NanoDrop, Wilmington,

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DE, USA).

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Oligonucleotide primers and probes

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The event-specific primers (G6H1-F/G6H1-R) and probe (qG6H1-p) were designed

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based on the 3’ end junction sequence of transgene insertion for G6H1 content

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quantification. Previously reported primer pair of SPS-F/SPS-R and SPS-P probe were

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used for quantification of rice endogenous gene saccharose phosphate syntheatase

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(SPS).21 All primers and probes were synthesized by Invitrogen Co., Ltd. Shanghai,



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China), and the sequence information was listed in Table 1 and Figure 1.

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Quantitative Real-time PCR

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Real-time PCR assays for the G6H1 event and SPS gene were optimized with a 25-uL

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reaction volume, containing 5 μL genomic DNA and 20 μL Real-time PCR Reaction

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Master Mix. The Real-time PCR Reaction Master Mix was consisted of the following

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reagents: 1 X PCR buffer, 200 μM dNTPs, 400 nM primers, 200 nM TaqMan probes,

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1.25 U Taq DNA polymerase (Huirui Biotech company, Shanghai, China). All real-time

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PCR reactions were carried out with the following program: 95°C for 10min, and then

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45 cycles of 94 °C for 15s and 60 °C for 60s. The fluorescent signal was monitored

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during each PCR cycle at the annealing step. Each quantitative PCR was repeated

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three times and each time with three replicate reactions.

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Collaborative Ring Trial

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The collaborative ring trial was organized by Chinese Academy of Agricultural

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Sciences (CCAAS). A total of 8 laboratories were invited and participated in the ring

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trial. Each laboratory received a kit for the validation study, including one tube of

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homozygous G6H1 genomic DNAs named with G1 (50 ng/μL, 100 μL), three bottle of

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G6H1 CRM powder samples (coded with S1, S2, and S3, respectively), one tube of 0.1

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X TE dilution buffer (5 mL in volume) and pre-mixed Real-time PCR Reaction Master

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Mix of G6H1 and SPS (2 tubes for each assay, and each tube with the volume of 2.0

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mL). Kits were shipped in dry ice to all laboratories. All participating laboratories also

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received a detailed protocol to follow and an excel file for reporting results. All

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laboratories were requested to report any deviation from the protocol which may


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have occurred during the preparation and execution of the real-time PCR

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experiments. The DNA extraction kit was not supplied, DNeasy 96 Plant Kit (Qiagen,

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Hilden, Germany) were recommend but no limited to each lab for blind samples DNA

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extraction. For each lab, the G6H1 genomic DNAs of G1 was requested to dilute to a

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series of ten-fold dilutions with the concentrations of 10.0, 1.0, 0.1, 0.01, and 0.001

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ng/μL (named from R1 to R5) using 0.1 X TE dilution buffer, and which were used to

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construct standard curves of G6H1 and SPS assays. For blind samples quantification,

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each lab was requested to randomly sample three sub-samples from each sample for

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DNA extraction and further quantitative analysis. The GM G6H1 content of each blind

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sample was determined by the average values of three sub-samples. The extracted

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DNAs of blind samples should be diluted to the final concentration of 5 ng/μL for

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real-time PCR amplification. During each PCR reaction, the negative and blank control

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reactions should be performed.

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Data Analysis

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In this ring trial, all participants were requested to return their results sheet within

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two months after receiving the trial package. Based on the returned results, PCR

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efficiency, linearity of regression, precision, and trueness were analyzed and

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calculated according to ISO 5725-2 and definition of minimum performance

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requirements for analytical methods of GMO testing reported by European Network

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of GMO Laboratories (ENGL).27, 30 The identified outlying results and/or laboratories

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were excluded from the analysis, in accordance with ISO 5725-2.30 The measurement

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uncertainty of G6H1 method was estimated according to the guidance document

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issued by the European Commission Joint research Center, Institute for Reference



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Materials and Measurements (JRC-IRMM).31 Statistical analysis of result differences

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among participating laboratories, such as least significant difference (LSD) test or the

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Q test, were performed using SPSS 13.0 software.


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Results and Discussion

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Establishment of G6H1 event-specific real-time PCR method

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For developing the event-specific method for G6H1, four sets of primers and probe

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were designed and pre-evaluated according to their specificity and sensitivity. The

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pre-evaluated results showed that the real-time PCR assay with primer pair (G6H1-F/

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G6H1-R) and probe (G6H1-P) presented one 90-bp amplicon in length could be used

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for further establishing the event-specific method of G6H1 (Data not shown). The

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sequences and locations of primer pair (G6H1-F/ G6H1-R) and probe (G6H1-P)with

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were shown in Figure 1.

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Specificity test. The specificity of G6H1 real-time PCR method was tested among

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different GM rice events (TT51-1, T1C-19, Kemingdao, and KF6) and GM plant species

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(maize, soybean, canola, and cotton). The amplified results showed that only positive

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fluorescent signal and traditional amplified curves were observed in the reactions

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with G6H1 DNA template, no fluorescent signal and amplified curves were observed

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in the reactions of mixed GM maize of MON810, MON863, and Bt176, mixed GM

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cotton MON531 and MON88913, mixed GM soybean of GTS40-3-2, and MON89788,

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mixed GM canola of Ms1Rsf1, Ms3Rf83, and GT73, non-GM rice Xiushui 110, and no

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template control (NTC) (As shown in Figure 2a). The results revealed the high

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specificity of developed G6H1 event-specific assay among different GM events.

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Construction of Standard Curve. To quantify the GM content of unknown samples,

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the standard curve of G6H1 real-time PCR assay was constructed employing five

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concentrations of diluted G6H1 genomic DNA (50.0, 5.0, 0.5, 0.05, and 0.005

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ng/reaction). High PCR efficiency of 100.95% was obtained (Figure 2b). The linearity

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between the logarithm of DNA copy numbers and Ct values was evaluated by R2



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values. The R2 value of 0.9999 was obtained (Figure 2b). The high PCR efficiencies

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and good linearity indicated that the standard curve is suitable for further

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quantitative measurement of GM rice with a large dynamic range.

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Sensitivity evaluation. To determine the lowest amount of the initial template DNA

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copies that could be reliably detected and quantified with 95% confidence level (i.e.,

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absolute limit of detection, LOD; limit of quantification, LOQ), four concentrations of

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genomic DNA samples (100, 50, 10, and 5 copies/reaction) were prepared and tested.

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Each sample were tested with three repeats, and each repeat with 20 parallel

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reactions. The expected positive fluorescent signals were obtained from all four

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concentrations in all replicated reactions. However, a high relative SD (RSD) value

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(49.81%) of genomic DNA amount was observed when using five haploid genomic

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DNA copies as template (Table 2). Therefore, the LOD value of five haploid genomic

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DNA copies was determined. The bias values of quantified results increased as the

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DNA amounts decrease, and a large bias of 38.54% was observed in the reactions of

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five haploid genomic DNA copies. To be quantified reliably, the approximately 10

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initial copies were required, and the LOQ was determined with 10 copies of haploid

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genome.

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Validation of G6H1 event-specific real-time PCR method

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In order to transfer the developed G6H1 method to other labs, one collaborative ring

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trial of the G6H1 real-time PCR assay was designed and organized according to the

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ISO 5725:1994. 26, 29 Since the SPS real-time PCR assay had been already validated for

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rice genomic DNA detection and quantification,21 we focused more on the validation

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of G6H1 real-time PCR assay in this ring trial, such as the PCR efficiency, linearity,


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precision, trueness, and measurement uncertainty.

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PCR efficiency and linearity. All participants were requested to prepare the calibrate

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DNA solutions (R1-R5) by gradient dilution from sample G1, and run the quantitative

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real-time PCR. Each reaction should be performed with three replicates and three

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repeats. The returned Ct values of three repeats were plotted against the log

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transformation of the copy number of the diluted solutions to obtain the standard

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curves, respectively (Figure 3). The PCR efficiency, linearity of regression, and slope

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were calculated from the standard curve to examine the applicability of quantitative

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real-time PCR assays.27 All the data showed high PCR efficiency, good linearity, and

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acceptable slope value in the curves for the G6H1 and SPS real-time PCR assays

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(Table 3). The key parameters of PCR efficiency, slope and the regression coefficients

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(R2) for the G6H1 and SPS assays from 8 laboratories were all in the acceptable range

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based on the requirements proposed by the European Network of GMO Laboratories

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(ENGL) guidelines.27 The linearity from each lab was higher than 0.9826, and the

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mean square regression correlations (R2) were 0.9989 and 0.9991 for both G6H1 and

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SPS real-time PCR assays, respectively. Meanwhile, the values of slope for the G6H1

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assay and the SPS assay from all labs were ranged from -3.24 to -3.58. The average

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values of slope for G6H1 assay (-3.43) and SPS assay (-3.40) were very similar,

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indicating no difference existed between these two assays in quantification even the

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target amplicons were completely different. The PCR efficiency was calculated using

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the following equation E= (10(-1/slope) -1) X 100%. As is shown in Table 3, the mean

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PCR efficiency was 95.55% for the G6H1 assay and 97.13% for the SPS assay. The

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results demonstrated good linearity and high PCR efficiency of G6H1 and SPS

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real-time PCR assays.



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Quantification of blind samples. In order to evaluate the accuracy of the G6H1

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real-time PCR method, three parameters including the trueness, precision, and

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measure uncertainty were calculated through the verification. Three G6H1 CRMs

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with the property values of 4.983%, 0.997%, and 0.499% (mass/mass ratio) were

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used as blind samples with the code of S1, S2, and S3 in this validation, respectively.

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In each participated laboratory, the S1-S3 blind samples was quantified according to

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the constructed standard curves, and the G6H1 content were calculated based on the

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formula of GM%= (G6H1 gene copy number/ SPS gene copy number) X100%. Each

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lab reported three values of G6H1 contents for each sample, and which were

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quantified from three sub-samples individually. The statistical data about GM content

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of three samples was exhibited in Table 4. The calculated values of GM content were

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checked with Dixon’s test and Grubbs’ test to find out remarkable outliers according

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to IS0 5725.29 No outliers was discovered from both the test.

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Trueness. The calculated GM G6H1 content of each sample was presented in Table 5.

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No significant outliers were excluded from all the returned data. The average test

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values of GM content for S1, S2, and S3 were 5.03%, 1.09%, and 0.53%, respectively.

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The bias between the quantified values and true values of three samples from all

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eight laboratories were ranged from 0.74% to 20.24% and shown in Figure 4. The

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average bias between the quantified values and true values of three samples among

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eight laboratories was within the range from 0.74% to 9.33%. In terms of real-time

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PCR method acceptance standard enacted by ENGL (within±25%), the G6H1

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event-specific method showed an ideal and reliable trueness.

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Precision. In accordance with ISO5725-2, the precision of a test method is mainly

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evaluated according to its reproducibility and repeatability. Both repeatability and


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reproducibility are usually reported as relative standard deviation. For instance, the

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relative repeatability (RSDr) describing the intra-laboratory variation, and relative

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reproducibility (RSDR) describing the inter-laboratory variation. The repeatability and

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reproducibility of the quantification result of each sample were estimated and listed

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in Table 4. The RSDr values ranged from 3.91% to 6.33% and RSDR values ranged from

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5.68% to 7.89% in blind samples analysis. All of these values were below the method

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acceptance criteria value of 25%, indicating that the G6H1 real-time PCR method was

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very reliable and repeatable even by different operators and equipment in different

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labs.

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Measurement uncertainty of quantified results. The measurement uncertainty (MU)

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of G6H1 method in blind samples quantification was also evaluated. Based on the

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guideline of measurement uncertainty for GMO testing laboratories, 30 the MU is

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calculated using the formulas of = + ( × ) and = 2 × . Herein,

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uc is the standard uncertainty, u0 is absolute standard uncertainty, RSU is relative

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standard uncertainty, c is the mean concentration of tested sample, and U is

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expanded uncertainty. The measurement uncertainty of the tested results is usually

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reported as an expanded uncertainty (U) under the 95% confidence level. The u0 and

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RSU was calculated by plotting the reproducibility standard deviation values (SDR)

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against the mean concentration (c) of the blind samples in the ring trial (Figure 5). u0

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is same with the absolute value of intercept of the linear regression (u0= 0.000044),

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RSU value is equal to the slope of the linear regression (RSU=0.056728). For the three

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tested blind samples, the expanded uncertainty (U) were calculated with the value of

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0.57%, 0.12% and 0.06% for S1, S2 and S3 sample, respectively. As a result, the

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measurement concentrations of the three samples were (5.03±0.57)% for S1,



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(1.09±0.12)% for S2, and (0.53±0.06)% for S3, respectively.

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Conclusion

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In summary, the G6H1 event-specific real-time PCR method was successfully

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established for accurate quantification of G6H1 contents with high specificity. The

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LOD and LOQ was determined with the value of 5 and 10 haploid genomic DNA

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copies, respectively. For the purpose of standardization, the developed G6H1

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event-specific real-time PCR method was well validated through a collaborative ring

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trial of 8 laboratories. The PCR efficiency, wide quantitative dynamic range, and good

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PCR performance in blind samples quantification indicated that the developed

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real-time PCR method is suitable for application in identification and quantification of

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GM rice event G6H1 and its derived products, and could be used in routine lab

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analysis in the future.


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Acknowledgements

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We heartily thank the following collaborators for their participation in this ring trial:

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GMOs and Derived Products Inspection and Supervision Center (Tianjin), Ministry of

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Agriculture, P.R. China ; Inspection Test Center for Environmental Safety of Transgenic

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Crops, (Jinan), Ministry of Agriculture, P.R. China; Inspection Test Center for

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Environmental Safety of Transgenic Crops, (Guangzhou), China; Inspection Test

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Center for Environmental Safety of Transgenic Crops, (Anyang), China; Chinese

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Academy of Inspection and Quarantine, China; National Institute of Metrology, China;

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Shanghai Entry-Exit Inspection and Quarantine Bureau, China; Cereal Quality

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Supervision and Testing Center, (Haerbin), Ministry of Agriculture, P.R. China. This

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work was supported by the National Transgenic Plant Special Fund

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(2016ZX08012-003 and 2016ZX08012-002), and the National Natural Science

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Foundation of China (31471670).



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17. Morisset, D.; Štebih, D.; Milavec, M.; Gruden, K.; Žel, J. Quantitative analysis of

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food and feed samples with droplet digital PCR. PLOS One. 2013, 8(5), e62583.

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18. Dobnik, D.; Spilsberg, B.; Košir, A. B.; Holst-Jensen, A.; Žel, J. Multiplex

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quantification of 12 European Union authorized genetically modified maize lines

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with droplet digital polymerase chain reaction. Anal. Chem. 2015, 87(16),

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19. Košir, A. B.; Spilsberg, B.; Holst-Jensen, A.; Žel, J.; Dobnik, D. Development and

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inter-laboratory assessment of droplet digital PCR assays for multiplex

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quantification of 15 genetically modified soybean lines. Sci. Rep. 2017, 7(1), 8601.

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20. Meng, YN.; Liu, X.; Wang, S.; Zhang, DB.; Yang, LT. Applicability of plasmid

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calibrant pTC1507 in quantification of TC1507 maize: an interlaboratory study. J.

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Agric. Food Chem. 2012, 60(1), 23-28.

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21. Jiang, LX.; Yang, LT.; Zhang, HB.; Guo, JC.; Mazzara, M.; Eede, G. V. D. International

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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(9), 3525-3532.

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22. Wu, YH.; Yang, LT.; Cao, YL.; Song, GW.; Shen, P.; Zhang, DB; Wu, G. Collaborative

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validation 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(25),

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5953-5960.

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23. Wei, JJ.; Li, FW.; Guo, JC.; Li, X.; Xu, JF.; Wu, G.; Zhang, DB.; Yang, LT. Collaborative

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ring trial of the papaya endogenous reference gene and its polymerase chain

364

reaction assays for genetically modified organism analysis. J. Agric. Food Chem.

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2013, 61(47), 11363-11370.

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24. Jacchia, S.; Nardini, E.; Bassani, N.; Savini, C.; Shim, J. H.; Trijatmiko, K.

367

International ring trial for the validation of an event-specific golden rice 2

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quantitative real-time polymerase chain reaction method. J. Agric. Food Chem.

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2015, 63 (20), 4954-4965.

370

25. Wei, JJ.; Le, HY.; Pan, AH.; Xu, JF.; Li, FW.; Li, X.; Quan, S.; Guo, JC.; Yang, LT.

371

Collaborative trial for the validation of event-specific PCR detection methods of


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genetically modified papaya Huanong No.1. Food Chem. 2016, 194, 20-25. 26. Horwitz, W. Protocol for the design, conduct and interpretation of method performance studies. Pure Appl. Chem. 1995, 67, 331-343. 27. Definition of minimum performance requirements for analytical methods of GMO

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testing-European

Network

of

GMO

Laboratories

(ENGL).

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http://gmo-crl.jrc.ec.europa.eu/doc/Min_Perf_Requirements_Analytical_method

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s.pdf.

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28. Yang, Y.; Li, L.; Yang, H.; Li, XY.; Zhang, XJ.; Xu, JF.; Zhang, DB.; Jin, WJ.; Yang, LT.

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Development of Certified Matrix-Based Reference Material as a Calibrator for

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Genetically Modified Rice G6H1 Analysis. J. Agric. Food Chem. 2018. 66 (14),

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3708-3715.

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29. International Organization for Standardization. ISO 21571:2005, Foodstuffs-

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Methods of analysis for the detection of genetically modified organisms and

385

derived products-Nucleic acid extraction. 2005.

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30. Institution, B. S. Accuracy (trueness and precision) of measurement methods and

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results -- part 2: basic method for the determination of repeatability and

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reproducibility of a standard measurement method. 1994.

389 390

31. Trapman, S.; Burns, M.; Broll, H.; Macarthur, R.; Wood, R.; Zel, J. Guidance document on measurement uncertainty for GMO testing laboratories. 2007.



391

Figure captions

392

Figure 1. The 3’ end event-specific sequence of GM rice event G6H1. The italic capital

393

letters were the exogenous DNA sequence of G6H1, and the capital letters were rice

394

genome DNA sequence. The designed real-time PCR primers were labeled with

395

underline, and the probe was labeled with red color and square frame.

396 397

Figure 2. The specificity test and construction of standard curve of G6H1

398

event-specific real-time PCR method. a) The real-time PCR amplified curves from

399

different GM rice events, GM maize, GM soybean, GM cotton, and GM canola

400

samples, etc. b) Real-time PCR amplified curves and constructed standard curve of

401

G6H1 method.

402 403

Figure 3. Standard curves of G6H1 event-specific and rice SPS real-time PCR assays

404

employing serially diluted solutions of R1-R5 in each laboratory. For each laboratory,

405

three standard curves were obtained from three individual repeat for each real-time

406

PCR assay.

407 408

Figure 4. Relative deviation from the true value of three blind samples for all

409

participating laboratories.

410 411

Figure 5. Linear regression produced by plotting mean measurement concentration

412

(c) against reproducibility standard deviation (SDR).


Page 20 of 29

Page 21 of 29


413

Tables

414

Table 1. Primers and Probes used for G6H1 real-time PCR method Amplicon size PCR assay

Name

Sequence (5’-3’) (bp)

SPS

G6H1

SPS-F

TTGCGCCTGAACGGATAT

SPS-R

CGGTTGATCTTTTCGGGATG

SPS-P

FAM-TCCGAGCCGTCCGTGCGTC-BHQ

G6H1-F

AAGCGTCAATTTGTTTACACC

G6H1-R

TCGATCTGCTGCAGCTTG

G6H1-P

FAM-ATGGATGTATCGCCACCAGCACC-BHQ1

415


81

90


416

Page 22 of 29

Table 2. The LOD and LOQ evaluation of G6H1 real-time PCR method using diluted calibrators Mean Ct values

DNA amount

Amount (copies) Bias (%)

SD

RSD

102.12

2.12

6.43

6.29

42.78

46.84

6.31

4.26

9.09

9.41

7.68

8.65

13.47

0.89

10.34

1.68

4.68

3.07

38.54

1.53

49.81

(copies)

Rep 1

Rep 2

Rep 3

Rep 1

Rep 2

Rep 3

Mean

100.00

33.59

33.48

33.41

95.46

103.08

108.24

50.00

34.48

34.61

34.74

51.29

46.84

10.00

36.98

36.91

37.20

8.96

5.00

38.25

39.38

37.91

3.69


Page 23 of 29


417

Table 3. Slope, PCR efficiency, and R2 values of the standard curves Lab

A

B

C

D

E

F

G

H

Rep

G6H1

SPS

slope

PCR efficiency (%)

Linearity (R )

slope

PCR efficiency (%)

Linearity (R2)

1

-3.41

96.45

0.9998

-3.42

96.06

0.9999

2

-3.40

96.84

0.9995

-3.36

98.44

0.9989

3

-3.46

94.54

0.9993

-3.40

96.84

0.9999

1

-3.45

94.92

1.0000

-3.36

98.44

1.0000

2

-3.44

95.30

0.9999

-3.29

101.35

0.9997

3

-3.45

94.92

1.0000

-3.56

90.94

0.9958

1

-3.49

93.43

0.9998

-3.31

100.50

0.9999

2

-3.39

97.24

0.9999

-3.35

98.84

0.9998

3

-3.41

96.45

0.9994

-3.24

103.54

0.9992

1

-3.41

96.45

0.9999

-3.42

96.06

0.9995

2

-3.41

96.45

0.9996

-3.40

96.84

0.9999

3

-3.38

97.63

0.9999

-3.45

94.92

0.9995

1

-3.48

93.80

0.9988

-3.38

97.63

0.9998

2

-3.47

94.17

0.9997

-3.38

97.63

0.9997

3

-3.35

98.84

0.9999

-3.38

97.63

0.9998

1

-3.58

90.25

0.9993

-3.55

91.29

0.9997

2

-3.51

92.71

0.9987

-3.54

91.64

0.9994

3

-3.48

93.80

0.9996

-3.45

94.92

0.9980

1

-3.33

99.66

0.9826

-3.40

96.84

0.9995

2

-3.48

93.80

1.000

-3.25

103.09

1.0000

3

-3.36

98.44

0.9994

-3.40

96.84

0.9920

1

-3.42

96.06

0.9998

-3.42

96.06

0.9998

2

-3.40

96.84

0.9994

-3.37

98.03

0.9988

3

-3.47

94.17

0.9993

-3.40

96.84

0.9999

-3.43

95.55

0.9989

-3.40

97.13

0.9991

Mean

2

418



419

Page 24 of 29

Table 4. Summary of validation results for the G6H1 event-Specific method Expected value Blind samples S1 (4.983%)

S2 (0.997%)

S3 (0.499%)

Labs returning results

8

8

8

Samples per lab

3

3

3

Number of outliers

0

0

0

Reason for exclusion

/

/

/

Mean value

5.02%

1.09%

0.53%

Repeatability SD

0.20%

0.04%

0.03%

Repeatability RSD

3.91%

3.45%

6.33%

Reproducibility SD

0.29%

0.06%

0.04%

Reproducibility RSD

5.68%

5.79%

7.89%

Bias (absolute value)

0.04%

0.09%

0.03%

Bias

0.74%

9.33%

6.21%

420


Page 25 of 29

421


Table 5. Determined GM% values of three blind samples from eight laboratories S1 (4.983%)

S2 (0.997%)

S3 (0.499%)

Lab Sub sample 1

Sub sample 2

Sub sample 3

Sub sample 1

Sub sample 2

Sub sample 3

Sub sample 1

Sub sample 2

Sub sample 3

A

4.72%

4.75%

4.56%

1.14%

1.23%

1.24%

0.57%

0.56%

0.49%

B

5.27%

4.90%

4.59%

1.02%

1.03%

1.05%

0.47%

0.44%

0.49%

C

5.36%

5.43%

5.81%

1.15%

1.13%

1.05%

0.61%

0.56%

0.52%

D

5.11%

4.57%

4.65%

1.11%

1.18%

1.15%

0.51%

0.58%

0.48%

E

5.04%

4.75%

4.61%

1.13%

1.08%

1.07%

0.55%

0.61%

0.62%

F

5.02%

5.26%

4.97%

1.01%

0.98%

1.04%

0.60%

0.57%

0.56%

G

5.25%

5.26%

5.13%

1.14%

1.06%

1.03%

0.47%

0.52%

0.51%

H

5.20%

5.05%

5.33%

1.07%

1.02%

1.06%

0.53%

0.52%

0.49%



422

Figures

423

Figure 1

424 425 426 427

Figure 2.

428 429


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430

Figure 3.

431 432



433

Figure 4

434 435 436

Figure 5

437


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TOC

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Development and Inter-laboratories Validation of Event-Specific

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