Ever since the first genetically modified (GM) crop was commercially planted, genetically modified organisms (GMOs) have come under suspicion from governments and citizens because of potential safety risks[@b1][@b2]. Many countries have stipulated legislation to regulate GMOs and GMO-derived products. The core of GMO regulation involves detecting GMOs, analyzing legality of their components in a particular region, and determining the need for labeling. Thus GMO detection technology is the requisite of GMO safety management.

Polymerase chain reaction (PCR) is the most generally accepted GMO detection technique. This is largely because of its ability to amplify specific DNA fragments from highly processed materials[@b2]. PCR-based GMO detection strategies include element screening, construct-specific, and transgenic event-specific methods[@b3]. The construct-specific detection method involves targeting the junction between two elements, and it is not able to distinguish two different events transformed with the same plasmid[@b4]. Event-specific detection can precisely distinguish legitimate transgenic events from related illegal varieties transformed with similar or identical transgenic constructs, thus it is often used to evaluate the legality of a GMO sample[@b3]. The screening method targets the most frequently used elements in transgenic constructs, has the lowest specificity, and is mainly used for rapid evaluation of high numbers of GMOs. In 2013, 336 GM crop varieties from 27 different species were commercialized worldwide[@b5]. This number is rising as ever more GM crops in the research stage enter field trials, in which, only partial varieties were developed event-specific detection methods. In general, it is not feasible to conduct PCR tests for all possible events during GMO detection. A common practice is to begin with general screening of a small number of targets common to numerous events, such as the *Cauliflower mosaic virus* (CaMV) 35S RNA gene promoter (P35S) and the terminator of the *Agrobacterium tumefaciens* Ti plasmid nopaline synthase gene (TNOS)[@b6][@b7][@b8][@b9].

Using the available GMO transformation information from the GM Crop Database (<http://cera-gmc.org/index.php?action=gm_crop_database>), we conducted preliminary statistical analyses into the presence of P35S and TNOS in GM crops. This revealed that 65.7% (67/102) of approved commercial GM events contain the P35S promoter, 53.49% (55/102) the NOS terminator, and 81.4 (83/102) either or both of these in their transgene constructs ([Supplementary Table S1](#s1){ref-type="supplementary-material"} online). In commercially important transgenic crops, the presence percentage of these two components is higher. Among the 28 commercial events of transgenic maize (*Zea mays*), only one (LY038; Monsanto, St. Louis, MO, USA) contained neither element, and in 16 commercial GMOs of rapeseed (*Brassica napus*) there were only two exceptions. Some transgenic events, such as GM maize Bt11, T25, and GM cotton Mon531 contained two copies of P35S in their transforming constructs. Consequently, the P35S promoter and NOS terminator are the most widely used GMO screening targets.

Because of the importance of the P35S promoter in screening detection of GMOs, a large variety of GMO screening tests have been established and published[@b10][@b11][@b12][@b13][@b14][@b15][@b16][@b17][@b18][@b19][@b20][@b21][@b22][@b23][@b24][@b25][@b26][@b27][@b28][@b29][@b30][@b31][@b32][@b33][@b34][@b35][@b36][@b37][@b38][@b39][@b40][@b41][@b42][@b43][@b44][@b45]. Some of these methods have been adopted by ISO, EU, China, and other countries and regions as standard methods for GMO detection[@b10][@b11][@b13][@b14][@b15][@b16][@b28][@b31][@b32][@b33][@b34][@b45]. The GMO Detection Method Database (GMDD) developed by Shanghai Jiao Tung University collected 37 methods from literature, ISO standards, and Chinese standards. These include 21 qualitative methods, 14 quantitative methods, and two methods used for microarray analysis (<http://gmdd.shgmo.org/>). In the GMDD, some primer pairs are simultaneously used by both qualitative and quantitative assays, and some methods are actually repeats of the same method using different primer names. The P35S promoter is one of the most frequently modified elements in GMOs. The P35S promoter sequences in different GMOs and vectors may be different from each other owing to origination from different strains or from modification in vector construction or mutation during the breeding process[@b13]. Previous oligonucleotide comparison revealed differences among P35S promoter sequences from the CaMV genome, event 176, Bt11, T25, MON810, and DLL25[@b17]. Morisett *et al*. identified a single nucleotide polymorphism (SNP) in the primer binding site of the P35S sequence in TC1507 maize, leading to inefficient amplification of testing primer/probe sets[@b9][@b46].

Although many P35S-based methods are available for the testing laboratories, only partial methods have gone through necessary validation processes and inter-laboratory studies against a small number of transgenic events[@b13][@b14][@b19][@b24][@b31][@b34][@b43][@b45]. Indeed, no one method has been systematically verified for accuracy and sensitivity across all commercially available transgenic events. The International Life Science Institute (ILSI) petitioned over 100 laboratories to survey the use of P35S and TNOS for the detection of GMOs. Some laboratories encountered methodological flaws of P35S in their testing, such as low sensitivity, low reproducibility, and false positives or negatives[@b9]. Holden *et al*. tested the suitability of five published P35S-based methods with eight maize reference materials, demonstrating that two methods had the flaws of poor linear regression parameters and multiple PCR amplicons in some of the testing materials[@b9].

The human immunodeficiency virus (HIV) often generates drug resistance mutations owing to highly variable gene and drug selection pressure. During detection of HIV-1 drug resistance mutations, the genotyping assay, combining reverse transcription PCR with sequencing technology or high resolution melting (HRM) analysis, is commonly used to detect all the possible mutations in HIV genome[@b47][@b48], the design of RT-PCR primers should target conserved regions flanking mutational hot spots, and the used primers must be specific to the region of interest. Similarly, the primers for GMO detection also should lie within conserved regions and be specific to the target sequence, and the detection of P35S requires amplification of a conserved region across different transgene events. Otherwise, the P35S-based methods would exhibit the above flaws, even result in false testing results during GMO screening.

P35S-based methodologies play an important role during the GMO screening phase. Currently, GMO detection laboratories select methods from different sources, including published literature, ISO standards, databases, or in-house developed methods. This heterogeneity in methodology may result in divergent test results if the P35S sequence carried by the testing sample was altered during construction of the transforming vector or in the breeding process. The ILSI survey revealed that most participating laboratories were interested in adopting a standardized method, which could generate consistent testing results and lead to better inter-laboratory reproducibility[@b9]. Currently, no optimal method is available that is based on the comprehensive comparison of the existing methods.

The purpose of this study was (1) to isolate the P35S sequence from different transgenic events, (2) to analyze methodological flaws in existing P35S-based detection methods using sequence alignment between primers/probes and the P35S sequences from different GMOs and constructs, and to confirm the defects found by the experiments, and (3) to design a general qualitative and quantitative detection system that targets the conserved region of P35S for high-coverage GMO screening.

Results
=======

Sequence alignment
------------------

A total of 67 GM events containing P35S have been collected by the GM crop database (<http://www.cera-gmc.org/GMCropDatabase>), of those, 23 unique GM events were available in the present study. After performing isolation of P35S promoters, nineteen P35S sequences were isolated from the following 16 transgenic events: GM soybean GTS-40-3-2, A5547-127; GM maize MON863, NK603, TC1507, Bt11, MON810, T25; GM cotton MON15985, MON88913, MON531, LLCotton 25, MON1445; GM rapeseed OXY235; and GM rice Kefeng 6 and KMD. The isolated sequences were submitted to the GenBank database, and accession numbers and sequences are summarized in [Table 1](#t1){ref-type="table"}. The P35S sequence length varied among the transgenic events, ranging from 307 bp in MON 810 to 1385 bp in LLCotton 25. Three transgenic events (Bt11, T25, and MON531) had two copies of P35S in their transforming constructs, with the sequence of these two copies different from each other for each event ([Table 1](#t1){ref-type="table"}).

Twelve P35S promoter fragments were collected: four maize events MON88017, MON89034, 59122, and 98140 (carrying three copies of P35S) from the patent database[@b49][@b50][@b51][@b52]; two rapeseed events Topas 19/2 and T45 from the application dossier; one rice event LLRice62 and three commonly used transgenic binary vectors pBI121, pCambia-1381, and pMCG161 from the GenBank database ([Table 1](#t1){ref-type="table"}). Both isolated and downloaded P35S sequences were aligned with the whole genome of CaMV (NC_001497.1) using the bl2seq program in NCBI, to determine the relative position of the P35S promoters in the CaMV genome. The homologous region and the SNP number of each P35S compared to CaMV genome are given in [Table 1](#t1){ref-type="table"}. In transgenic events selected for this study, all of the isolated P35S promoters normally drive the target genes to express functional proteins. We therefore concluded that all P35S sequences were complete. In accordance with the results of the sequence alignment, we obtained the conserved region of P35S across different transgenic events, corresponding to the genomic region of CaMV between positions 7148 and 7342. The sequence alignment results revealed that 13 P35S sequences, from events A5547-127, NK603, MON810, T25, 59122, MON88017, 98140, MON15985, MON88913, MON531 and LLRice62, exhibited 100% identity with the CaMV genomic sequence, while the other P35S sequences exhibited different degrees of variation ([Table 1](#t1){ref-type="table"}). For the conserved regions, 13 P35S sequences from GTS-40-3-2, MON863, TC1507, Bt11 (two copies), MON89034, MON531, LLcotton 25, MON1445, Kefeng 6, KMD, pCambia-1381, and pMCG161 showed differences from the CaMV genomic sequence, with homologies from 93% to 99%, the other collected P35S sequences showed 100% identity with the CaMV genomic sequence ([Table 1](#t1){ref-type="table"}). The sequence comparison also revealed that seven P35S promoters, from NK603, MON88017, MON15985, MON88913, MON531, Kefeng 6, and pCambia-1381, had duplicated enhancer regions; these were defined as double enhancer promoters ([Table 1](#t1){ref-type="table"}).

Twenty-four different detection methods targeting the P35S promoter were identified from published papers and detection standards, and labeled as M1 to M24 ([Table 2](#t2){ref-type="table"}). Of these, the M1 method was adopted by ISO 21569[@b15], the M13 method by ISO 21570[@b32], three methods (M1, M14, and M11) by the National Standards of China[@b11][@b28], four (M1, M2, M15, and M16) by the Industrial Standards of China[@b10][@b16][@b35], and four (M1, M4, M12, and M14) were collected by the EU Database of Reference Methods for GMO Analysis (<http://gmo-crl.jrc.ec.europa.eu/gmomethods/>)[@b14][@b31][@b34][@b45]. These 24 methods included 10 conventional qualitative PCR methods and 14 real-time PCR methods. In addition, partial primer pairs for real-time PCR methods, such as M1, M3, M4, M5, M12, and M14, were simultaneously used for conventional qualitative detection ([Table 2](#t2){ref-type="table"}). Primer and probe sequences are given in [Table 2](#t2){ref-type="table"}. Primer pairs (or primer/probe sets) were aligned with the whole genome of CaMV using the bl2seq program, and their positions shown in [Table 2](#t2){ref-type="table"}. According to the positions of primers/probes in the CaMV genome, we observed that with the exception of five methods (M2, M7, M10, M12, and M18) the reverse primers of 19 methods were located outside of the conserved region of P35S. The P35S sequences from the CaMV genome, multiple transgenic events, and binary vectors with SNPs, together with primer/probe sets, were aligned using AlignX in the Vector NTI 9 software suite. Sequence alignment revealed that the vast majority of these 19 methods contained SNPs in the binding site of primer (SNPs were also shown in [Table 2](#t2){ref-type="table"}), and their reverse primers mismatched the P35S of MON88913 cotton, 98140 maize and the P35S regulating Pat gene of T25 maize ([Supplementary Fig. S1a-f](#s1){ref-type="supplementary-material"} online). Both the forward and the reverse primers of method M10 located outside the conserved region, mismatching the P35S of GTS-40-3-2, MON863, MON810, MON89034, and MON1445; and the forward primer of M10 located outside the conserved region, mismatching the P35S of NK603, Bt11, MON88017, MON15985, MON88913, MON531, Kefeng6, KMD, OXY235 and pCambia-1381 ([Supplementary Fig S1g](#s1){ref-type="supplementary-material"} online). The forward primer of M18 did not match the P35S of GTS-40-3-2, MON863, MON89034, and MON1445 ([Supplementary Fig S1h](#s1){ref-type="supplementary-material"} online). Sequence alignment revealed that 21 of the 24 published P35S-based methods had defects, resulting in missed detection of partial transgenic events; the exceptions were M2, M7, and M12, where both primers and probes were located within the conserved region of P35S.

To investigate the sequence consistency of primer binding sites for methods M2, M7, and M12, the P35S conserved region from the CaMV genome, multiple transgenic events, and binary vectors harboring nucleotide alterations, together with primer/probe sets, were aligned ([Fig. 1](#f1){ref-type="fig"}). For the M2 method, a SNP in TC1507 maize located at the binding site of the forward primer, and a SNP in Bt 11 at the binding site of the probe. For the M7 method, the binding site of the probe was a high variability region containing four SNP mutations, resulting in a mismatch with most of the transgenic events and vectors. Furthermore, the SNP in TC1507 also existed in the binding site of the reverse primer, corresponding to the second nucleotide of the 3′ end of the reverse primer. For the M12 method, two SNPs in TC1507 and Bt11 were both situated in the binding site of the forward primer, with the SNP in TC1507 corresponding to the third nucleotide of the 3′ end of the primer. We speculated that the nucleotide mutation in the primer binding sites would cause inefficient amplification of methods M2, M7, and M12, and that the M7 probe located in a highly variable region could give rise to an abnormal fluorescent signal when detecting mutated P35S targets.

Qualitative detection of P35S in GM crops using the collected methods
---------------------------------------------------------------------

The 24 collected P35S-based methods were used to detect P35S in MON88913. Since at least one primer lied outside the P35S region of MON88913, nineteen primer pairs failed to detect the P35S target when MON88913 genomic DNA was used as template; while five methods (M2, M7, M10,M12 and M18) successfully detected the P35S target ([Fig. 2a](#f2){ref-type="fig"}). Sequence comparison indicated that the above 19 methods would also fail to detect P35S in 98140 maize, whereas, this was unable to be confirmed due to unavailable to 98140 maize ([Supplementary Fig. S1a--f](#s1){ref-type="supplementary-material"} online). The M10 method was used to amplify P35S fragments from 23 GM varieties, including GM soybean GTS40-3-2, A5547-127, A2704-12; GM maize Bt11, TC1507, T25, Bt176, NK603, MON89034, M88017, MON810, MON863, 59122; GM cotton MON88913, MON1445, MON531, LLcotton25, MON15985; GM rapeseed T45, Topas19/2, OXY235; and GM rice Kefeng 6, and KMD. The 188-bp amplicon was not visualized in 15 of the samples: GTS40-3-2, Bt11, Bt176, NK603, MON89034, MON88017, MON810, MON863, MON88913, MON1445, MON531, MON15985, OXY235, Kefeng6, and KMD ([Fig. 2b](#f2){ref-type="fig"}). The 23 GM varieties tested above were also analyzed for the existence of P35S by the M18 method. Of those, four GM crops (GTS-40-3-2, MON89034, MON863, and MON1445) were failed to yield an expected 196-bp PCR fragment ([Fig. 2c](#f2){ref-type="fig"}). The expected product was observed in MON531 cotton when using the M18 method, because two copies of P35S are present in MON531, with one copy having completely matched primer binding sites for M18 ([Supplementary Fig. S1h](#s1){ref-type="supplementary-material"} online). The detection results for P35S in GM crops were in agreement with the above sequence alignment results. The qualitative detection of P35S demonstrated that most existing P35S-based methods had flaws resulting in missed detection of partial GM crops harboring P35S.

Influence of SNP mutations on PCR performance of the M2, M7 and M12 methods
---------------------------------------------------------------------------

The binding sites of the primer/probe set contained SNPs for the M2, M7, and M12 methods ([Fig. 1](#f1){ref-type="fig"}). To evaluate the effect of SNP mutations on the PCR performance of these three methods, a series of dilutions of extracted DNAs from MON810, TC1507, and Bt11 events and the binary vector pMCG161 were used as calibrators to set up standard curves, with each of the five dilutions assayed in triplicate. According to the sequence alignment results, the DNA from MON810 contained no SNPs, and could therefore be used as a control; DNA from events Bt11, TC 1507 and plasmid pMCG161 was used to assess the influence of the primer/probe mismatch in the M2, M7, and M12 methods; The amplification plots and corresponding standard curves are shown in [Fig. 3](#f3){ref-type="fig"}, the R^2^ and slope data of the standard curves are summarized in [Table 3](#t3){ref-type="table"}, and the Ct values listed in [Supplementary Table S2](#s1){ref-type="supplementary-material"} online. The characteristic parameters of the standard curves constructed using MON810 were in the acceptable range for all three methods, with a slope range from −3.108 to −3.282, and R^2^ values ranging from 0.994 to 0.997[@b53]. The amplification plot of Bt11 showed obvious gradient changes among serial dilutions for the M2 and M12 methods, but the standard curve had a very shallow slope (−1.967 for M2, −1.965 for M12), exceeding the acceptable range (−3.1 to −3.6). No gradient change and poor repeatability among the three parallel reactions were visualized using Bt11 with the M7 method, resulting in standard curves with a poor correlation coefficient (0.72) and extreme slope (−1.657). The TaqMan assays for the M2 and M7 methods with TC1507 showed extreme slopes and abnormal amplification curves, similar to that seen when Bt11 was assayed by M7; for the M12 method, the TaqMan assays with TC1507 also exhibited an abnormal slope (−1.864), but obvious gradient changes among the serial dilutions and good repeatability among parallel reactions were observed. Partial dilutions of pMCG161 were assayed using the three methods, the characteristic parameters of the standard curves were in the acceptable range for methods M2 and M12, whereas, the M7 method generated poor fluorescent signals, and relatively large Ct values.

The TaqMan assays revealed that the SNP mutation in TC1507 generated large anomalies in the PCR performance of the M2, M7, and M12 methods, in agreement with the previous study[@b9]. The pMCG161 plasmid had two additional nucleotides and two SNP at the binding site of the M7 probe compared to the other events ([Fig. 1](#f1){ref-type="fig"}), leading to very poor fluorescent amplification curves. For the Bt11 event, two SNP sites at the M7 probe binding site severely affected the PCR performance of the M7 method, one SNP at the M2 probe binding site and the M12 forward primer binding site only resulted in larger Ct values and a shallower slope. In conclusion, the mismatch between primer/probe and DNA template influenced the amplification plot and the characteristic parameters of the standard curves.

Primer/probe design
-------------------

A fragment of approximately 195 bp between position 7148 and 7342 in the CaMV genome was relatively conserved across different P35S promoters, even though it contained multiple SNPs dividing it into smaller discrete segments. This conserved fragment was present in all collected P35S sequences in this study, furthermore, two copies were contained in double enhancer promoters such as the enhanced P35S promoter in pCambia-1381, maize NK603 and MON88017, cotton MON15985, MON88913 and MON531, and rice Kefeng6. Multiple candidate primer/probe sets were designed to anneal to conserved segments carrying no SNP. To select the best primer/probe set, all possible primer and probe combinations were tested for amplicon size and specificity using 0.1 ng genomic DNA from transgenic maize TC1507 as a template, which possesses a P35S promoter and has been observed to have inefficient amplification in some of the methods. The most effective, reliable, and robust primer/probe set was 35SEF/35SER/35SEP, yielding a 125 bp amplicon, and labeled as M25 ([Table 2](#t2){ref-type="table"}). The binding sites of the selected primers/probe had 100% identity to all of the tested P35S promoter fragments ([Fig. 1](#f1){ref-type="fig"}).

Conventional PCR detection of the P35S promoter
-----------------------------------------------

The amplification stability of the primer pair 35SEF/35SER was tested using the genomic DNA from the 23 GMOs described above. Electrophoresis revealed that the unique 125 bp fragment was amplified from all samples containing a P35S promoter ([Fig. 4a](#f4){ref-type="fig"}). Specificity testing revealed that no amplification occurred in samples lacking the promoter (data not shown). Therefore, conventional PCR amplification using the primer pair 35SEF/35SER reliably and specifically detected the P35S promoter in GM crops.

In practice, DNA extracted from GM food or feed tends to be highly degraded or very low in quantity. To evaluate the detection sensitivity of the new qualitative detection system, genomic DNA from five transformants (TC1507, GTS-40-3-2, MON1445, KMD, and OXY235) containing the P35S promoter, representing the five major transgenic crops (soybean, maize, rapeseed, rice, and cotton), were serially diluted to 100, 50, 20, and 10 copies per microliter and used as templates for PCR analysis. The lowest detectable template quantity required for maize TC1507 was estimated to be 20 copies; for soybean GTS40-3-2, 50 copies; for cotton MON1445, 20 copies; for rice KMD, 20 copies; and for rapeseed OXY235, 50 copies ([Fig. 4b](#f4){ref-type="fig"}). These differences in PCR results may be because of different DNA organization. These results indicated that the detection sensitivity of our new qualitative method was as low as 50 copies or fewer in the tested species.

Performance of P35S quantitative method on different GM crops
-------------------------------------------------------------

Variations in the DNA templates of different crops may affect test results during GMO screening by P35S-targeted methods. To evaluate the suitability of the newly developed P35S-based quantitative method for detecting various GM crops, the five events (TC1507, GTS-40-3-2, MON1445, KMD, and OXY235) were subjected to real-time PCR to analyze the performance of the quantitative PCR. The conserved regions in GTS-40-3-2 soybean, TC1507 maize, KMD rice, and MON1445 cotton all have SNPs present, while OXY235 rapeseed does not. A series of dilutions for each of the extracted DNAs was made, corresponding to 50000, 5000, 500, 50, and 10 copies/µL for events GTS-40-3-2, OXY235, TC1507, and KMD, and to 28000, 5000, 500, 50, and 10 copies/µL for event MON1445. The serial dilutions were used as calibrators to establish standard curves for P35S detection, and each dilution was assayed in triplicate. Standard curves were created by plotting Ct values against the logarithm of transgene copy numbers, good agreement was observed between the quantity of template and the Ct values for each event ([Fig. 5](#f5){ref-type="fig"}). The square regression coefficients (R^2^), slope, and amplification efficiency are summarized in [Table 4](#t4){ref-type="table"}; these meet the minimum performance requirements for analytical methods of GMO testing defined by the European Network of GMO Laboratories (ENGL)[@b53]. The R^2^ values ranged from 0.997 for TC1507 to 1.000 for GTS-40-3-2, this is significantly higher than the ENGL minimum requirement of 0.98. The slopes across the five events ranged from −3.411 in GTS-40-2 to −3.225 in KMD, this is within the acceptable range of −3.6 ≤ slope ≤ −3.1. Based on the slope of the standard curve, the efficiency of this P35S PCR method was estimated to be from 96.4% to 104.2%, close to the ideal efficiency of 100%. The real-time PCR assays verified that SNPs in conserved regions did not influence the amplification efficiency of the newly developed quantitative method. We therefore conclude that our real-time assay is suitable for quantifying the P35S promoter copy number in various GM crops.

To determine the limits of detection (LOD) and quantification (LOQ) of the real-time PCR method, five genomic DNA templates (TC1507, GTS-40-3-2, MON1445, KMD, and OXY235) from the five major crops were each diluted to 80, 50, 40, 20, 10, 5, and 1 copies/µL to perform real-time PCR assays in 10 replicates. The discrepancy in the Ct values across the ten replicates became larger with decreased template copy number ([Table 5](#t5){ref-type="table"}). All 10 PCR replicates had typical fluorescence amplification curves for the five tested GMOs when the template copy number increased to 10, whereas only partial reactions were positive when using five or one copy template dilutions. There were no visual differences in PCR performance across the different GM crops. Therefore, the LOD of our quantitative PCR method reached 10 copies for the different GM crops. The relative standard deviation (RSD) of Ct values among the 10 replicates did not exceed 25% with the reduction of template copy number; we speculate that the LOQ of our method was approximately 50 copies, based on the relationship between LOQ and LOD elucidated by the guidance document of the Joint Research Centre, Institute for Reference Materials and Measurements (JRC-IRMM)[@b54].

Discussion
==========

Screening methods are often used directly to make a preliminary judgment on whether or not samples are GMOs. Precise and accurate detection methods are a prerequisite for reliable control of GMOs, and the screening method should be suitable for a wide range of GMOs. The survey on the GMO detection methods revealed that multiple qualitative or quantitative methods were developed and published for the same one target sequence, for instance, 24 methods targeting P35S were established, 14 methods targeting TNOS, 11 methods for *NPTII* gene, and 15 methods for *Bar* gene (<http://gmdd.shgmo.org/>). Method heterogeneity across testing laboratories can lead to adverse testing results during the screening phase of GMO detection, which can consequently cause problems for international trade. Due to the difficulty in detecting all the different transgene events, a single artificial sequence is recommended to be developed as a universal barcode, which would not be found in natural DNA sources. During the process of developing GMOs in future, all transgene developers could be required to include the single artificial sequence that would make detection of all transgene events easier by one method. However, the implementation of this idea needs to obtain approval and support of both transgene developers and regulators.

P35S are likely to remain an important component of GMO products at present and in the foreseeable future. Currently, 24 methods targeting different regions of the P35S sequence are reported and adopted by individual research and testing laboratories[@b10][@b11][@b12][@b13][@b14][@b15][@b16][@b17][@b18][@b19][@b20][@b21][@b22][@b23][@b24][@b25][@b26][@b27][@b28][@b29][@b30][@b31][@b32][@b33][@b34][@b35][@b36][@b37][@b38][@b39][@b40][@b41][@b42][@b43][@b44][@b45].This experiment demonstrated that all of the existing P35S screening systems had flaws, and that these may generate false negative results or/and underestimate GMO content during GMO detection. In this study, a conserved section in the enhancer region of P35S was identified by sequence alignment, and a general PCR method targeting this region was established. The validation results demonstrated that we have developed an improved general P35S screening system suitable for available transformants, whereas, we are still unable to test the method with all of the available commercial transgenic events. Sequence comparison revealed that many P35S sequences from transgenic events are different from each other. Because we do not have access to all existing approved GMOs that carry P35S, and unapproved GM materials are even more difficult to access, some nucleotide alterations may still be undiscovered in the P35S conserved region. While isolating the P35S sequences, we found that our DNA sequence data for the P35S region from GTS-40-3-2, TC1507, Bt11, T25, and MON1445 were inconsistent with the sequence information released by the GMDD database or described in US patent documents ([Table 1](#t1){ref-type="table"}). Therefore, it is important that testing laboratory staff pay attention to the sequence alterations of P35S introduced in transgenic events, check the homology of primer/probe sequences and templates, and to know the applicability of each method to any given sample; this will avoid detection errors during GMO screening.

Most existing GMO labeling systems are based on transgenic content, but not all transformants have a corresponding event-specific detection method. If a real-time PCR method targeting general transgenic components could be used for GMO quantitation, then GMOs without an event-specific detection method can be quantified and labeled. However, the new detection methods still have problems that require addressing because the copy number of the detecting target is not the same for different single copy transformation events. In this analysis the detection target locates in the enhancer region, which is usually reused in the double enhancer promoter to enhance regulatory activity. For instance, the NK603, MON88017, MON15985, MON88913, MON531, and Kefeng 6 events all contain a double enhancer promoter. Furthermore, events such as Bt11, T25, and MON531 contain two copies of P35S in their transgene constructs, and for the MON531 event one copy is a double enhancer promoter. Hence, GMO content can be overestimated when using the P35S method for events harboring a double enhancer promoter or multiple copies of P35S. Therefore, when this new quantitative PCR method is used to estimate the exogenous gene copy number of samples, DNA extracted from the same sample should be used to construct the standard curve. In addition, if only P35S shows positive signal for test samples in practice, a subsequent experiment detecting other genomic sequence of CaMV, should be performed to rule out CaMV itself as contaminant in the plant DNA samples. In conclusion, the use of this new P35S method, which covers a wide range of GMOs, will lead to more consistent results of GMO detection during the screening phase among different testing laboratories.

Methods
=======

Plant materials
---------------

Transgenic materials, including seed powder of transgenic soybean (10% GTS 40-3-2) and transgenic maize (10% 59122, 5% BT11, 5% BT176, 10% MON810, 10% MON863, 5% NK603, and 10% TC1507), were purchased from the Institute for Reference Materials and Measurements (IRMM, Geel, Belgium). Seed powder of transgenic maize (MON88017, MON89034), cotton (MON88913, MON1445, MON15985, and MON531), and leaf DNA of soybean (A5547-127), maize (T25), cotton (LLcotton25), and rapeseed (Topas 19/2, T45, and Oxy235) were purchased from the American Oil Chemists\' Society (Champaign--Urbana, IL, USA). Transgenic soybean (A2704-12), transgenic rice (Kefeng 6, KMD) and non-transgenic crop seeds were already available in our own laboratory.

DNA extraction
--------------

A DNeasy Plant Mini Kit (Qiagen, Hilden, Germany) was used to extract and purify genomic DNA from seeds or seed powders in accordance with manufacturer\'s instructions. DNA purity was checked using a NanoDrop 2000 spectrophotometer (Thermo Fisher, Waltham, MA, USA), and sample concentrations further quantified using a Versafluor Fluorometer (Bio-Rad, Hercules, CA, USA) with a Quanti-iT^TM^ PicoGreen® dsDNA Assay Kit (Invitrogen, Carlsbad, CA, USA).

Cloning of the P35S Promoter
----------------------------

Primers used for isolating the P35S sequences from different transgenic events were designed using Primer Premier 5.0 software (PREMIER Biosoft International, Palo Alto, CA) according to the nucleotide sequences flanking the P35S promoter. Primers were synthesized by Sangon (Shanghai, China); their sequences are given in [Supplementary Table S3](#s1){ref-type="supplementary-material"} online. The PCR samples were prepared using a KOD-Plus kit (Toyobo, Osaka, Japan) in a sample volume of 50 μL containing 20 ng of genomic DNA, 1× KOD-Plus PCR buffer, 200 μM of each dNTP, 1 mM MgSO4, 100 nM of each primer, and 0.5 units of KOD-Plus DNA polymerase. PCRs were performed on a C1000™ Thermal Cycler (Bio-Rad, Hercules, USA) using the following program: 94°C for 2 min (initial denaturation); 35 cycles of 94°C for 15 s (denaturation) and 68°C for 3 min (annealing and extension); and 68°C for 7 min (final extension). PCR products harboring a target band were recovered and subcloned into the pZErO-2 vector (Invitrogen, Carlsbad, CA, USA) via an *Eco*RV restriction enzyme site. Ligation products were transformed into *Escherichia coli* strain TOP10F (Invitrogen, Carlsbad, CA, USA), and positive clones screened. Plasmids containing the PCR products were sequenced using M13 forward and reverse primers (Tsingke, Beijing, China).

Sequence alignment
------------------

Pairwise comparison of nucleotide sequences was performed using the bl2seq program available from the National Center for Biotechnology Information (NCBI) (<http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch&BLAST_SPEC=blast2seq&LINK_LOC=align2seq>). Regions conserved in aligned P35S promoters were discovered based on the positions of different P35S promoters in the CaMV genome. The P35S region across the GMOs and binary vectors, together with the whole genome sequence of CaMV (NC_001497.1), were aligned using the AlignX program of the Vector NTI 9 software suite (Invitrogen).

Primers and probes
------------------

Oligonucleotide primers and TaqMan fluorescent dye-labeled probes were designed according to the conserved region of P35S promoters in the relevant GM crops using a specified optimal melting temperature of approximately 60°C for primers and 70°C for probes. The 5′ ends of probes were labeled with the fluorescent reporter 6-carboxy-fluorescein (FAM), and the 3′ ends with the minor groove binder non-fluorescent quencher (MGBNFQ). All primers and fluorescent probes were synthesized by Sangon Biotech (Shanghai, China).

PCR reactions
-------------

Conventional PCR was run on a Bio-Rad C1000™ Thermal Cycler using an optimized conventional PCR mixture: 1× PCR buffer (with 1.5 mM MgCl~2~), 200 μM of each dNTP, 0.25 μM of each primer, 1 U EX Taq™ (TaKaRa, Otsu, Japan), and 20 ng genomic DNA in a total volume of 20 µL. The PCR used the following cycle conditions: initial denaturation for 120 s at 94°C; 35 cycles of 30 s at 94°C, 30 s at 60°C, and 30 s at 72°C; and terminal elongation for 2 min at 72°C. PCR products were size fractionated using 2% agarose gel electrophoresis in 1× TAE buffer, and visualized with ethidium bromide. The UV-fluorescent emission was recorded with a Gel Doc XR system using Quantity One software (Bio-Rad).

Real-time PCR assays were carried out on a CFX96 Real-Time System (Bio-Rad) in a final volume of 20 µL. The reaction mixture for the P35S promoter contained 1× TaqMan Universal PCR Master Mix (Applied Biosystems, Foster City, CA, USA), 400 nM primers, and 200 nM probe. All real-time PCRs were performed using the same program: pre-digestion at 50°C for 2 min; initial denaturation and uracil-N-glycosylase deactivation at 95°C for 10 min; and 50 cycles of 15 s at 94°C (denaturation) and 1 min at 60°C (annealing and extension). Fluorescence was measured after each annealing and extension step using CFX Manager ver. 1.6 (Bio-Rad). Data analysis was performed using the CFX Manager ver. 1.6 (1.6.541.1028) software (Bio-Rad).

Author Contributions
====================

G.W. designed this experiment and wrote the main manuscript; Y.W. designed this experiment, wrote the manuscript, and prepared figures; Y. Wang., J.L. and W.L. performed this experiment; L.Z. and Y.L. advised on the experimental design and commented on the manuscript; other authors, including X.L., L.Z. and J.L., reviewed the manuscript.

Supplementary Material {#s1}
======================

###### Supplementary Information

Development of a general method for detection and quantification of the P35S promoter based on assessment of existing methods

This study was supported by a grant from the National Major Special Project for the Development of Transgenic Organisms of China (grant nos. 2014ZX08012-003, 2014ZX0801202B, and 2014ZX08012-005).

![Sequence alignment of the P35S conserved region from CaMV, transgenic events and binary vectors harboring SNPs, together with primer/probe sets of the M2, M7, M12 methods and primer/probe set (M25) designed in this study.](srep07358-f1){#f1}

![PCR amplification of P35S targets in GM crops using existing methods.\
(a) Amplification of the P35S fragment in MON 88913 event using the 24 existing P35S-based methods, Lanes 1--24 correspond to the M1-M24 methods. (b--c) Amplification of the P35S fragments in 23 transgenic events by methods M10 and M18, respectively. Lanes 1--24 correspond to the following samples: GM soybean (1) GTS40-3-2, (2) A5547-127, (3) A2704-12; GM maize (4) Bt11, (5) TC1507, (6) T25, (7) Bt176, (8) NK603, (9) MON89034, (10) M88017, (11) MON810, (12) MON863, (13) 59122; GM cotton (14) MON88913, (15) MON1445, (16) MON531, (17) LLcotton25, (18) MON15985; GM rapeseed (19) T45, (20) Topas19/2, (21) OXY235; GM rice (22) Kefeng 6, (23) KMD; (24) non-GM crop mixture (soybean, maize, cotton, rapeseed and rice).](srep07358-f2){#f2}

![TaqMan assays using M2, M7, and M12 methods with serial DNA dilutions from events MON810, TC1507, Bt11, and the vector pMCG161.\
Standard curves were constructed based on the amplification plot. MON810 maize, which does not contain a SNP, was used as the control. Methods M2, M7 and M12 were assessed with events TC1507, Bt11, and vector pMCG161.](srep07358-f3){#f3}

![Testing of the amplification stability and sensitivity of the qualitative PCR detection.\
(a) Amplification of P35S in different GM crops. Key: Lane M, DL 1000 DNA Marker, Lanes 1--24 correspond to the following samples: GM soybean (1) GTS40-3-2, (2) A5547-127, (3) A2704-12; GM maize (4) Bt11, (5) TC1507, (6) T25, (7) Bt176, (8) NK603, (9) MON89034, (10) M88017, (11) MON810, (12) MON863, (13) 59122; GM cotton (14) MON88913, (15) MON1445, (16) MON531, (17) LLcotton25, (18) MON15985; GM rapeseed (19) T45, (20) Topas19/2, (21) OXY235; GM rice (22) Kefeng 6, (23) KMD; (24) non-GM crop mixture (soybean, maize, cotton, rapeseed and rice). (b) Sensitivity of the qualitative PCR method. Serially diluted DNA extracts of maize TC1507, soybean GTS 40-3-2, cotton MON 1445, rice Kefeng 6, and rapeseed OXY235 were used as templates. Lanes 1--4 correspond to 100, 50, 20, and 10 haploid genome copies, respectively; each template was run with two parallel PCR reactions.](srep07358-f4){#f4}

![Amplification plot and standard curves for real-time quantitative PCR assays of P35S using serially diluted genomic DNA from five transgenic events as calibrators.\
(a--e) correspond to the amplification plots and standard curves of events GTS-40-3-2, MON1445, OXY235, KMD1, and TC1507, respectively.](srep07358-f5){#f5}

###### P35S sequence information from different transgenic events

                                                                         Identity of whole sequence   Identity of conserved region              
  ---------- ------------- ------ --------------- --------------------- ---------------------------- ------------------------------ ------ ---- ------------------------------------------------
  Soybean     GTS-40-3-2    322      KJ608131           7148-7468                   95%                            16                93%    13   Different from GMDD[b](#t1-fn2){ref-type="fn"}
               A5547-127    530      KJ608130           6908-7437                   100%                           0                 100%   0                           
  Maize         MON863      322      KJ608136           7148-7468                   95%                            16                93%    13                          
                 NK603      551      KJ608140      7090-7344,7090-7381              100%                           0                 100%   0                   Double enhancer
                TC1507      651      KJ608142           6913-7565                   99%                            6                 99%    1    Different from GMDD[c](#t1-fn2){ref-type="fn"}
                 Bt11       502      KJ608143           7072-7563                   95%                            26                94%    11   Different from GMDD[d](#t1-fn2){ref-type="fn"}
                            501      KJ608144           7072-7563                   95%                            28                94%    11                          
                MON810      307      KJ608135           7137-7443                   100%                           0                 100%   0                           
                  T25       530      KJ608147           6908-7437                   100%                           0                 99%    2                           
                            319      KJ608129           7027-7342                   98%                            5                 100%   0    Different from GMDD[e](#t1-fn2){ref-type="fn"}
                 59122      530    US20060070139        6908-7437                   100%                           0                 100%   0                           
               MON88017     615      US8212113     7090-7344,7090-7443              100%                           0                 100%   0                   Double enhancer
               MON89034     296      US8062840          7148-7443                   97%                            9                 95%    9                           
                 Bt176      203                         7365-7567                   98%                            4                  \\    \\                    Not complete
                 98140      436     US7897846B2         6909-7344                   100%                           0                 100%   0                           
                            438     US7897846B2         6909-7346                   100%                           0                 100%   0                           
                            438     US7897846B2         6909-7346                   100%                           0                 100%   0                           
  Cotton       MON15985     610      KJ608138      7090-7344,7090-7438              100%                           0                 100%   0                   Double enhancer
               MON88913     519      KJ608139      7090-7347,7090-7344              100%                           0                 100%   0                   Double enhancer
                MON531      615      KJ608145      7090-7344,7090-7443              100%                           0                 100%   0                   Double enhancer
                            322      KJ608146           7148-7468                   95%                            16                93%    13                          
                 LL25       1385     KJ608134           6105-7483                   94%                            78                95%    10                          
                MON1445     322      KJ608137           7148-7468                   95%                            16                93%    13   Different from GMDD[f](#t1-fn2){ref-type="fn"}
  Rapeseed      Oxy235      840      KJ608141      6495-6972,7090-7443              99%                            3                 100%   0                           
                  T45       530                         6908-7437                   99%                            1                 100%   0                           
              Topas 19/2    530                         6908-7437                   99%                            1                 100%   0                           
  Rice          Kefeng6     787      KJ608132      7017-7343,7018-7478              95%                            32                94%    11                  Double enhancer
                  KMD       462      KJ608133           7017-7478                   97%                            16                94%    11                          
               LLRice602    530      KF036176           6908-7437                   99%                            1                 100%   0                           
  Binary        pBI121      835      AF485783           6605-7439                   99%                            3                 100%   0                           
  vector      pCAMBIA1381   781     AF234302.1     7018-7445,7021-7345              96%                            17                94%    11                  Double enhancer
                pMCG161     1332    AY572837.1          6114-7440                   96%                            55                94%    12                          

^a^the accession number of CaMV is NC_001497.1 in the GenBank database.

^b,\ c,\ d,\ e,\ f^ represent isolated sequences different from the released sequences in the GMDD database, and correspond to the following urls: <http://gmdd.shgmo.org/sequence/view/1>, <http://gmdd.shgmo.org/sequence/view/10>, <http://gmdd.shgmo.org/sequence/view/39>, <http://gmdd.shgmo.org/sequence/view/17>, and <http://gmdd.shgmo.org/sequence/view/23>.

###### Primers and fluorescent probes used in the qualitative and real-time quantitative PCR systems to detect the P35S promoter

  No.    Orientation                    Name                    Sequence(5′-3′)[b](#t2-fn2){ref-type="fn"}[,c](#t2-fn3){ref-type="fn"}   Position[a](#t2-fn1){ref-type="fn"}   Amplicon                   Reference                      Note
  ----- ------------- ---------------------------------------- ------------------------------------------------------------------------ ------------------------------------- ---------- ------------------------------------------- ------------
  M1       forward                     35S-1                                           GC**T**CCTACAAATGCCATCA                                       7190--7208                  195      [@b10],[@b11],[@b12],[@b13],[@b14],[@b15]        
           reverse                     35S-2                                             GATAGTGGGATTGTGCGTCA                                        7365--7384                           [@b10],[@b11],[@b12],[@b13],[@b14],[@b15]        
            probe                     35S core                                      **TC**TCCACTGACGTAAGGGATGACGCA                                   7346--7371                                            [@b13]                          
  M2       forward                     35S-F3                                           CGACAGT**G**GTCCCAAAGA                                       7248--7265                   74                       [@b16]                          
           reverse                     35S-R3                                           AAGACGTGGTTGGAACGTCTTC                                       7300--7321                                            [@b16]                          
            probe                      35S-P                                        TGGACCCCCACCCACGAGGA**G**CATC                                    7266--7290                                            [@b16]                          
  M3       forward                   P35S 1-5′                                    ATTGATGTGA**T**A**TC**TCCACTGACGT                                  7334--7358                  101             [@b17],[@b18],[@b19],[@b20]               
           reverse                   P35S 2-3′                                        CCTCTCCAAATGAAATGAACTTCCT                                      7410--7434                                  [@b17],[@b18],[@b19],[@b20]               
            probe                     P35S-Taq                                   CCC**A**CTATCCTTCGCAAGACC**C**TTCCT                                 7376--7402                                            [@b18]                          
  M4       forward                       sF                                           CGTCTTCAAAGCAAGTGGAT**T**G                                     7316--7337                   79             [@b12],[@b13],[@b21],[@b22]               
           reverse                       sR                                             TCTTGCGAAGGATAGTGGGATT                                       7373--7394                                  [@b12],[@b13],[@b21],[@b22]               
            probe                    P-35S_3-P                                      **TC**TCCACTGACGTAAGGGATGACGCA                                   7346--7371                                         [@b13],[@b22]                      
  M5       forward                     35SFZ1                                        CCGACAGT**G**GTCCCAAAGATGGAC                                    7247--7270                  162                    [@b13],[@b23]                      
           reverse                     35SFZ2                                       ATATAGAGGAA**G**GGTCTTGCGAAGG                                    7384--7408                                         [@b13],[@b23]                      
            probe                     35S core                                      **TC**TCCACTGACGTAAGGGATGACGCA                                   7346--7371                                            [@b13]                          
  M6       forward                   P-35S_4-L                                          GACGTAAGGGATGACGCACAA                                        7354--7374                   81                       [@b24]                          
           reverse                   P-35S_4-R                                        CCTCTCCAAATGAAATGAACTTCCT                                      7410--7434                                            [@b24]                          
            probe                    P-35S_4-P                                    CCCACTAT**C**CTTCGCAAGACC**C**TTC                                  7376--7400                                            [@b24]                          
  M7       forward                35S-promoter.for                                    **GA**CATTGCGATAAAGGAAAGGC                                     7205--7226                   68                       [@b25]                          
           reverse                35S-promoter.rev                                     GGGTCCATCTTTGGGA**C**CA                                       7254--7272                                            [@b25]                          
            probe               35Spromoter-specific                            ATC**G**TT**G**AAGATGCCTCT**G**CCGACA                                7228--7252                                            [@b25]                          
  M8       forward                      35SF                                            CCTACAAATGCCATCATTGCG                                        7193--7213                  205                       [@b13]                          
           reverse                      35SR                                           **G**GGTCTTGCGAAGGATAGTG                                      7378--7397                                            [@b13]                          
            probe                     35S Wolf                                          CAAAGATGGACCCCCACCCACG                                       7260--7281                                            [@b13]                          
  M9       forward                     3-16f                                             CGTCTTCAAAGCAAGTGGAT                                        7316--7335                  105                       [@b26]                          
           reverse                     3-100r                                           GAA**G**GGTCTTGCGAAGGA                                       7383--7400                                            [@b26]                          
            probe                      3-67t                                               ACGCACAATCCCACTA                                          7367--7382                                            [@b26]                          
  M10      forward                   P-35S_31-L                                        AGACTGGCGAACAGTTCATACAGA                                      6956--6979                  188                    [@b27],[@b28]                 Microarray
           reverse                   P-35S_31-R                                          CAATGGAATCCGAGGAGGT                                         7124--7142                                         [@b27],[@b28]                      
            probe                    P-35S_31-P                                     TG**C**TCCACCA**T**GTTGACGAAG                                    7011--7031                                         [@b27],[@b28]                      
  M11      forward                    P-35S-AF                                           AAGATGCCTCTGCCGACAGT                                        7235--7254                  142                       [@b29]                     Microarray
           reverse                    P-35S-AR                                           GATTGTGCGTCATCCCTTAC                                        7357--7376                                            [@b29]                          
            probe                      P-35S                                        GAACGTCTTCTTTTTCCACGATGCTCCTCG                                   7280--7309                                            [@b29]                          
  M12      forward      P180-F(TM-35S-F[@b33],35S-FTM[@b31])                            GCCTCTGCCGACAGT**G**GT                                       7240--7257                   82         [@b12],[@b30],[@b31],[@b32],[@b33]            
           reverse      P180-R(TM-35S-R[@b33],35S-RTM[@b31])                            AAGACGTGGTTGGAACGTCTTC                                       7300--7321                              [@b12],[@b30],[@b31],[@b32],[@b33]            
            probe      P180-P(TM-35S-Pro[@b33],35S-TMP[@b31])                           CAAAGATGGACCCCCACCCACG                                       7260--7281                                  [@b30],[@b31],[@b32],[@b33]               
  M13      forward                    P-35S-F                                           GACGTAAGGGATGACGCACAA                                        7354--7374                   81                    [@b24],[@b32]                      
           reverse                    P-35S-R                                       **C**CTCTCCAAATGAAATGAACTTCCT                                    7410--7434                                         [@b24],[@b32]                      
            probe                     P-35S-P                                       CCC**A**CTATCCTTCGCAAGACCCTTCC                                   7376--7401                                         [@b24],[@b32]                      
  M14      forward                    p35S-cf3                                          CCACGTCTTCAAAGCAAGTGG                                        7313--7333                  123             [@b11],[@b12],[@b13],[@b34]               
           reverse                    p35S-cr4                                        TCCTCTCCAAATGAAATGAACTTCC                                      7411--7435                                  [@b11],[@b12],[@b13],[@b34]               
            probe                     35S core                                      **TC**TCCACTGACGTAAGGGATGACGCA                                   7346--7371                                            [@b13]                          
  M15      forward                     35S-F                                         GC**T**CCTACAAATGCCATCATTGC                                     7190--7212                  195                    [@b16],[@b35]                      
           reverse                     35S-R                                           GATAGTGGGATTGTGCGTCATCCC                                      7361--7384                                         [@b16],[@b35]                      
  M16      forward                     35S-F2                                         TCATCCCTTACGTCAGTGGA**G**                                      7347--7367                  165                       [@b10]                          
           reverse                     35S-R2                                           CCATCATTGCGATAAAGGAAA                                        7203--7223                                            [@b10]                          
  M17      forward                35SFZMP1(U-35S)                                     CCGACAGT**G**GTCCCAAAGATG                                      7247--7267                  158                    [@b36],[@b37]                      
           reverse                35SFZMP2(D-35S)                                     AGAGGAA**G**GGTCTTGCGAAGG                                      7384--7404                                         [@b36],[@b37]                      
  M18      forward                     SP1 F                                             TTGCTTTGAAGACGTGGTTG                                        7310--7329                  196                       [@b38]                          
           reverse                     SP1 R                                           ATTCCATTGCCCAGCTA**T**CT                                      7134--7153                                            [@b38]                          
  M19      forward                     35S3F                                           GCCATCATTGCGATAAAGGAAAGG                                      7202--7225                  173                       [@b39]                          
           reverse                     35S6R                                          TTGTGCGTCATCCCTTACGTCAGTG                                      7350--7374                                            [@b39]                          
  M20      forward                      CM01                                        **CA**CTACAAATGCCATCATTGCGATA                                    7192--7216                  220                       [@b40]                          
           reverse                      CM02                                        CTTATATAGAGGAA**G**GGTCTTGCGA                                    7387--7411                                            [@b40]                          
  M21      forward                     35S-A                                           AA**G**GGTCTTGCGAAGGATAG                                      7380--7399                  227                       [@b41]                          
           reverse                     35S-B                                         AGT**G**GAAAAGGAAGGTGGC**T**                                    7173--7192                                            [@b41]                          
  M22      forward                35S F(p35S- ar1)                                      CCTACAAATGCCATCATTGCG                                        7193--7213                  207                    [@b42],[@b43]                      
           reverse               35S R (p35S-af1u)                                     **G**GGTCTTGCGAAGGATAGTG                                      7378--7397                                         [@b42],[@b43]                      
  M23      forward                   35S-111 F                                         GT**G**GTCCCAAAGATGGACCC                                      7253--7272                  111                       [@b44]                          
           reverse                   35S-111 R                                      CCCTTACGTCAGTGGAGATATC**A**CA                                    7339--7363                                            [@b44]                          
  M24      forward                      35SA                                           AA**G**GGTCTTGCGAAGGATAG                                      7380--7399                  149                       [@b42]                     Nested PCR
           reverse                      35SB                                         AGT**G**GAAAAGGAAGGTGGC**T**                                    7173--7192                                            [@b42]                          
           reverse                      35SC                                           ACAGT**G**GTCCCAAAGATGGA                                      7250--7269                                            [@b42]                          
  M25      forward                     35SEF                                           CATCATTGCGATAAAGGAAAGGC                                       7204--7226                  125                     This study                        
           reverse                     35SER                                            TGCTTTGAAGACGTGGTTGGA                                        7308--7328                                                                            
            probe                      35SEP                                               TCGTGGGTGGGGGTC                                           7247--7268                                                                            

^a^Primer and probe positions are indicated relative to the genome sequence of CaMV (GenBank accession NC_001497.1).

^b^Probes of M1--M24 were labeled with 5′ FAM and 3′ BHQ1. The probe of M25 was labeled with 5′ FAM and 3′ MGBNFQ.

^c^Nucleotides mismatching the reference CaMV sequence or partial GMO templates, were highlighted in bold.

###### Parameters of standard curves for the M2, M7, and M12 methods

  Event      Parameter      M2       M7      M12
  --------- ------------ -------- -------- --------
  MON810     R^2^ value   0.994    0.997    0.996
               Slope      −3.169   −3.108   −3.282
  TC1507     R^2^ value   0.486    0.659    0.988
               Slope      −0.790   −1.797   −1.864
  Bt11       R^2^ value   0.980    0.720    0.965
               Slope      −1.967   −1.657   −1.965
  pMCG161    R^2^ value   0.998    0.504    0.998
               Slope      −3.288   −1.759   −3.175

###### Parameters of the P35S standard curves when using serially diluted genomic DNA from five events as calibrators

  Events        R^2^ value   Slope    Amplification efficiency
  ------------ ------------ -------- --------------------------
  GTS-40-3-2      1.000      −3.411            96.40%
  TC1507          0.997      −3.246           103.30%
  MON1445         0.998      −3.329            99.70%
  OXY235          0.999      −3.409            96.50%
  KMD             0.999      −3.226           104.20%

###### Estimation of LOD and LOQ of real-time PCR methods when using serially diluted genomic DNA from five events as templates

  Transgenic event    template copy no.   signal ratio   mean Ct value    SD    RSD
  ------------------ ------------------- -------------- --------------- ------ ------
  GTS-40-3-2                 80              10/10           34.63       0.46   1.32
                             50              10/10           35.11       0.31   0.89
                             40              10/10           35.51       0.47   1.33
                             20              10/10           36.28       0.31   0.86
                             10              10/10           37.33       0.49   1.31
                              5               8/10            \\          \\     \\
                              1               4/12            \\          \\     \\
  MON1445                    80              10/10           33.27       0.24   0.73
                             50              10/10           34.47       0.26   0.75
                             40              10/10           34.96       0.27   0.78
                             20              10/10           35.69       0.48   1.33
                             10              10/10           36.64       0.49   1.32
                              5               9/10            \\          \\     \\
                              1               4/12            \\          \\     \\
  OXY235                     80              10/10           33.34       0.26   0.79
                             50              10/10           34.47       0.26   0.75
                             40              10/10           35.16       0.47   1.33
                             20              10/10           36.80       0.39   1.07
                             10              10/10           38.29       0.58   1.52
                              5               2/10            \\          \\     \\
                              1               2/10            \\          \\     \\
  KMD                        80              10/10           33.28       0.65   1.94
                             50              10/10           34.40       0.37   1.08
                             40              10/10           35.09       0.32   0.93
                             20              10/10           36.50       0.49   1.34
                             10              10/10           37.15       0.80   2.14
                              5               3/10            \\          \\     \\
                              1               5/10            \\          \\     \\
  TC1507                     80              10/10           32.40       0.38   1.18
                             50              10/10           33.31       0.36   1.08
                             40              10/10           34.03       0.44   1.30
                             20              10/10           35.73       0.40   1.13
                             10              10/10           36.17       0.57   1.59
                              5               2/10            \\          \\     \\
                              1               5/12            \\          \\     \\

[^1]: These authors contributed equally to this work.
