A New Reference Plasmid “pGMT27” Provides an Efficient Transgenic Detection Method for Flue-Cured Tobacco

Owing to the economic value of its foliage, tobacco (Nicotiana tabacum) is cultivated all across the world. For the detection of genetically modified (GM) tobacco, there is a lack of universal standard material which ultimately limits the detection methods because the accuracy and comparability of the results cannot be ensured. Here, we prepared a reference plasmid “pGMT27” for the detection of GM tobacco, which was 18,296 bp in length harboring two of the tobacco endogenous and seven exogenous genes. By using qualitative PCR test for the nine genes, 10 copies were used for plasmid sensitivity. In the quantitative real-time PCR (qPCR) assays with pGMT27 as a calibrator, the reaction efficiencies for P-35S and NR were 101.427% and 98.036%, respectively, whereas the limit of detection (LOD) and limit of quantification (LOQ) were 5 copies and 10 copies per reaction. For standard deviation (SD) and relative standard deviation (RSD) of the Ct values, the repeatability values were from 0.04 to 0.42 and from 0.18% to 1.29%, respectively; and the reproducibility values were from 0.04 to 0.39 and from 0.18% to 1.14%, respectively. For the unknown sample test, the average conversion factor (Cf) was 0.39, and the accuracy bias was from −15.55% to 1.93%; for precision, the SD values ranged from 0.02 to 0.62, while RSD values were from 1.34% to 10.6%. We concluded that using the pGMT27 plasmid as a calibrator provided a highly efficient transgenic detection method for flue-cured tobacco.


Introduction
Tobacco is one of the most widely cultivated nonfood crops and has emerged as an extensively investigated model plant with the accelerated development of molecular biology research. Attributable to its agronomic importance, tobacco is grown in over 125 countries for its foliage, mainly consumed as cigarettes, cigars, and snuff. According to the FAO (Food and Agriculture Organization) data, approximately 4 million hectares of lands are used for cultivating tobacco, one-third of which is in China alone [1]. As smoking is health related [2][3][4], consumers appear to have more cautious attitudes to tobacco than other crops and GM tobacco, as well as other GM crops.
ough applications from biotechnology to tobacco products have been limited, unintentional release of GM tobacco seeds and leaves of nonpermissible sort to the market is possible [5]. Now, GM composition detection in tobacco is becoming an international trade barrier for monitoring the presence of adventitious GM tobacco. So, when entering into the market, it is indispensable for the tobacco companies to enhance controls on import and export of the tobacco product. In many countries, respecting public's right to information and education, the GMO threshold levels of food and feed products have already been established [6][7][8], Consequently, to ensure the accurate and comparable measurements, selection of standard material for GMO detection becomes critical [9]. e standard reference material includes matrix and plasmid. Plasmid has many advantages, such as ease of availability, straightforward manipulation, repeatable production, and low cost [10]. Some plasmids as calibrators have been used for transgenic soybean, maize, cotton, and other crops, including ERM-AD413, ERM-AD415, ERM-AD427, and ERM-AD425. ey are also used to calibrate the transgenic maize MON810, NK603, 98140, and transgenic soybean 356043 [11]. However, the lack of standard material has limited the GM tobacco detection methods. Up until now, no standard material as the calibrator for detecting GM tobacco has been found, and no corresponding products have been reported. In the tobacco trade market, tobacco is usually sold in the form of tobacco product, including flue-cured tobacco, redried leaf tobacco, and cigarettes. Here, we reported a plasmid for the GM tobacco test, which was designated as pGMT27. It was constructed based on plant expression vector pBI121. pGMT27 contained two tobacco endogenous genes (actin and NR), genetic elements of the P-35S promoter, T-NOS terminators, selective marker genes of NPT II, HPT, Bar, and aadA, and GUS reporter gene. ese seven exogenous genes could make the screening rate of transgenic tobacco reach 100% theoretically in our previous screening strategy [12]. Accordingly, a qualitative PCR detection method for detecting the nine targets was developed. Furthermore, a qPCR assay for P-35S and NR was also developed by using plasmid DNA as the calibrator. ereby, we concluded that using the pGMT27 plasmid as a calibrator provided a highly efficient transgenic detection method for flue-cured tobacco.

Flue-Cured Tobacco and Genomic DNA Isolation.
Transgenic flue-cured tobacco tested by the FAPAS (Food Analysis Performance Assessment Scheme) and CORESTA (Cooperation Centre for Scientific Research Relative to Tobacco) proficiency test in 2014 was defined as 100% positive GM with P-35S and T-NOS. Nontransgenic fluecured tobacco was collected in our laboratory. All materials were leaves' flour. Plant genome extraction kit (TIANGEN Biotech, China) was employed to extract DNA according to the manufacturer's protocol. Total DNA quantification was performed using a NanoDrop ND-2000 Spectrophotometer (NanoDrop Technologies, USA), OD260/ OD280 ≈ 1.80-1.90, and it was diluted to a 100 ng/μL working stock with sterile distilled water, which was stored at −80°C.

Construction of the pGMT27
Plasmid. According to the screening strategy previously reported [12], we synthesized a 3,818 bp fragment of HPT + Bar + aadA + actin + NR junction and subcloned it into pMD18-T simple vector by restriction enzyme sites, EcoR I and Sac I. Furthermore, the full-length fragment of five targets was digested with EcoR I/ Sac I and cloned into the plant expression vector pBI121 instead of "T-NOS" with the same enzyme sites, so as to create the recombinant pGMT27 plasmid, which includes nine target DNA fragments consisting of seven exogenous genes and two endogenous. en, pGMT27 plasmid was isolated and purified by using TIANGEN Plasmid Miniprep Kit (TIANGEN Biotech, China) according to the manufacturer's guidelines. Afterwards, it was identified by Sac II and EcoR I restriction digests and DNA sequencing (Shanghai Generay, China). Total DNA quantification was performed on a Qubit® 2.0 Fluorometer (Invitrogen Life Technologies, USA) by using the Qubit® dsDNA BR Assay Kit (Invitrogen Life Technologies, USA) following the manufacturer's directions. e copy numbers were calculated on the basis of pGMT27 plasmid size (18,296 bp) by using Avogadro's constant (6.023 × 10 23 ). e plasmid DNA molecular weight (660 g/mol/dp) was calculated with formula (A) proposed by Lee et al. [13].

Qualitative PCR Assay for the pGMT27 Plasmid.
For the LOD test of the qualitative PCR, the plasmid copy number was prepared by 0.1 × TE buffer (10 mM Tris, 0.1 mM EDTA, and pH 8.0). All samples were prepared by serially diluting 10 6 copies/μL to 1 copy/μL, and all the reactions were performed in 20 μL volume containing 1.0 U of rTaq DNA polymerase (TAKARA, Japan) and 2 μL of 10 × PCR buffer along with 125 μM dNTP, 0.5 μM of primers, and 1 μL of plasmid DNA. PCR amplifications were performed in C1000 Touch PCR (Bio-Rad, USA) with the program as follows: one step of 5 min at 95°C; 40 cycles of 30 s at 94°C, 45 s at 63-65°C (T-NOS annealing temperature was 63°C, and the others were 65°C), and 45 s at 72°C; and 5 min at 72°C. Amplification products were electrophoresed in 2% agarose gels for approximately 40 min at 100 V. Primers were synthesized and purified by Shanghai Generay Biotech Co., Ltd. (Table S1). Real-Time PCR System (Invitrogen Life Technologies, USA), and the data were analyzed by ViiA 7 software v1.2. PCR primer and probes for P-35S and NR were synthesized by Shanghai Generay Biotech Co., Ltd. (Table S2).

Dilution for Standard Curves, Reproducibility, Repeatability, and Sensitivity.
To test the availability of pGMT27, the plasmid was prepared as a calibrator and was serially diluted to 10 6 , 10 5 , 10 4 , 10 3 , and 10 2 copies/μL with 0.1 × TE buffer to construct the standard curves. For the repeatability and reproducibility test, the same five dilution points were also used. Each dilution point was amplified in three replicates. For the reproducibility test, the amplification was finished in three different days. For sensitivity, it included LOD and LOQ, and the pGMT27 plasmid was diluted to 50, 25, 10, and 5 copies/μL with 0.1 × TE buffer.

Conversion Factor (Cf ) Calculation and Unknown
Samples' Test. In order to minimize the differences between plasmid DNA and plant genomic DNA in PCR efficiencies, calculating the Cf value is a prerequisite [14]. For the unknown samples' test, 100% transgenic flue-cured tobacco DNA was diluted into a gradient of 100%, 50%, 25%, 12.5%, and 6.25% (V/V) with the content of 0% transgenic fluecured tobacco DNA, reactions were run in ViiA TM 7 Real-Time PCR System, and then target gene and endogenous gene copy number were calculated according to the pGMT27 standard curve. e calculations were performed according to formula (B) suggested by Pi et al. [15], and the final Cf value represented the mean of five calculated Cfs. e unknown sample GM content (%) was calculated by using formula (C) according to Pi et al. [15]. Each reaction was repeated thrice. (2)

Construction of the pGMT27 Plasmid and LOD Test by
Qualitative PCR. Figure 1 represents a plasmid pGMT27 (18,296 bp), constructed in this study for the qualitative detection of GM tobacco. It harbored nine targets including seven exogenous and two endogenous targets shown in Table 1. Sac II and EcoR I restriction digests ( Figure S1) and DNA sequencing (data not given) validated the acquisition of expected plasmids. For testing sensitivity in the qualitative PCR, nine targets by using 10 6 , 10 5 , 10 4 , 10 3 , 10 2 , 50, 10, 5, and 1 copies of the pGMT27 plasmid as templates were measured by the LOD method. Nine targets were detected as low as 5 copies, which meant that the lowest testing level was 5 copies (Figure 2). However, we recommend that the template should be more than 10 copies if pGMT27 is a positive control plasmid in the practice test work because some groups have no amplification signals in the three repeats when only 5 copies of the templates were present (data not shown); therefore, the uncertainty is obviously greater because of the low template amount.

Preparation of the pGMT27
Plasmid as the Calibrator. e pGMT27 plasmid was diluted to 10 6 , 10 5 , 10 4 , 10 3 , and 10 2 copies/μL, so as to establish standard curves for P-35S and NR targets for qPCR. Standard curves of the two reactions are displayed in Figure 3. e correlation coefficient (R 2 ) values of the standard curves were 0.997 and 0.998 for both P-35S and NR which indicated excellent linearity. PCR efficiencies were 101.427% and 98.036%, respectively, and data were generated by equation E � 10 −1/slope − 1 [16] and analyzed by ViiA 7 software v1.2, and standard curves of the two targets showed excellent linearity.

Repeatability, Reproducibility, LOD, and LOQ for qPCR
Assays. For repeatability and reproducibility of Ct from P-35S and NR, targets of the pGMT27 plasmid were diluted and amplified for one day and three different days, respectively, and the data are shown and analyzed in Table 2. For the repeatability test, the SD of the Ct values ranged from 0.04 to 0.42, and the RSD was less than 1.29%. For the reproducibility test, the SD and RSD values were in the range of 0.04 to 0.39 and 0.18% to 1.14%, respectively. For qPCR assays, usually, repeatability of RSD less than 25% and reproducibility below 35% were accepted according to the European Network of GMO Laboratories (ENGL) guideline in 2015 [17], and our results indicated good repeatability and reproducibility, which were all in the acceptable range. To test the LOD and LOQ of plasmid pGMT27 in the qPCR, four kinds of concentrations (50, 25, 10, and 5 copies/μL) were prepared. According to ENGL [17], the acceptance criterion of LOD and LOQ should be less than 25 copies and 50 copies, respectively. In the assays, the results determined that the LOD and LOQ were 5 copies and 10 copies per reaction for P-35S and NR, respectively (Table 3).

Estimation of Cf Values and Quantitative Analysis of Unknown Samples.
In order to analyze the GM tobacco content of unknown samples by using the pGMT27 plasmid as a calibrator in qPCR, the Cf values were calculated by the copy ratios of P-35S to NR. e endogenous gene NR was a single-copy gene in the tobacco genome [5]. In Table 4, Cf values for each time, including three parallel reactions, were 0.34, 0.39, 0.40, 0.40, and 0.41, and the mean value of the Cfs was 0.39. e SD and RSD values of the Cf were 0.03 and 6.77%, respectively, which were all in the acceptable range. With the aim of testing the GM content of the unknown   Journal of Food Quality samples, six concentrations were used for the analysis, and these samples were 0.5%, 2%, 5%, 6.25%, 12.5%, and 25% (v/ v), which were prepared by mixing the samples of several GM and non-GM flue-cured tobacco DNA. GM percentages of unknown samples were calculated by formula (C). e qPCR analysis is shown in Table 5, where the accuracy of bias ranged from −15.55% to 1.93%; however, our bias values were lower than the acceptance criteria (−25% to 25%) according to ENGL [17]. Precision of SD and RSD ranged from 0.02 to 0.62 and from 1.34% to 10.6%. All these results indicated good repeatability, which were all in the acceptable range.

Discussion
On a global scale, the number of genetically engineered plants has risen exponentially in recent years, adding up to the complexity of efforts taken by the enforcement laboratories to detect not only the authorized GMOs in food and feed samples but also the unauthorized ones. In order to produce reliable data, research laboratories need to harmonize their techniques to quantify GMOs or to demonstrate that different methods used for the quantification of GMOs are commutable [6,7]. Quantitative examination of GMOs is usually carried out by the determination of the   amount of event-specific target with respect to the reference gene [8]. In the current study, seven exogenous ones were frequent, and the theoretical coverage reached 100% in the tobacco genetic transformation event [12]. Approaches for the detection and quantification of many event-specific targets of commercially available genetically modified organisms and reference genes are available. Additionally, there are many studies that demonstrate the development of methods and their in-house validation [6,10]. Other studies indicated that the LOD was 5 and 20 copies, respectively, by using the plasmid as a reference molecule for detecting transgenic canola and soybean [18,19]; correspondingly, in our study, nine targets were detected as low as 5 copies, which meant that the lowest testing level was 5 copies. Nonetheless, it is important to critically evaluate any validation status before implementing a method in the research laboratory for routine sample analyses. For reliable quantification of GMOs, laboratories need appropriate organization and quality management systems, and several critical points should be considered in the analytical procedures, such as sampling, sample preparation, and DNA extraction, which have been described recently [10]. Differential qPCR for the detection of unauthorized GMOs is based on the occurrence of several common elements in different GMOs (e.g., promoter and genes of interest). A statistical model was developed to study the difference between the number of targets of such a common sequence and the number of event-specific targets that can identify the approved GMO and the donor organism of the common sequence. When this difference statistically deviates from zero, the presence of an unauthorized GMO can be deduced [20]. However, this approach has low sensitivity, and it is reliable only if the presence of an unknown GMO exceeds 30%. Furthermore, a qPCR assay for P-35S and NR was also developed by using plasmid DNA as the calibrator. e R 2 values of the standard curves were 0.997 and 0.998 for both P-35S and NR, those R 2 values ≥ 0.98 were accepted [9], and other researchers' results ranged from 0.995 to 0.999 [18,[21][22][23]. eoretically, the Cf score should be 1.0 for the quantitative PCR test when the exogenous gene and endogenous gene are both one copy [5,15], but in this study, the Cf value was 0.39, which is far more below 1.0, so we could observe three potential reasons: (1) large differences between plasmid DNA and plant genomic DNA in quantitative PCR efficiencies, and some studies involving crops validate this as Cf values ranging from 0.53 to 0.83 have been reported [14,21,23]; (2) these samples' GM content was defined as 100% positive by the FAPAS, but the fact was there was less than 100% GM content; (3) flue-cured tobacco DNA partially degrades after curing [24]. e SD and RSD values of the Cf were 0.03 and 6.77%, respectively, which were all in the acceptable range. e advancement of new approaches that are suitable for the quantitative measurement of specific sequences is a promising solution that can overcome the drawbacks of the currently used ways/methods. In the past, several alternative methods for the detection of GMOs were developed that explored different aspects of amplification, detection, and    Journal of Food Quality identification approaches [25,26]. Recently, additional methods have been developed [27][28][29][30][31]. Consequently, in the current study, a qualitative PCR detection method for detecting the nine targets was developed. Highly linear and efficient reactions indicated that the standard curves of the pGMT27 plasmid were suitable for further quantitative test [32]. Meanwhile, using the pGMT27 plasmid as a calibrator provided a highly efficient method in the transgenic detection of flue-cured tobacco.

Conclusion
e newly developed screened plasmid pGMT27 provides better coverage of the GM elements that could be present in a sample and will facilitate advancements in the detection of unauthorized/unknown GM tobacco. It includes two tobacco endogenous genes (actin and NR), exogenous gene of the P-35S promoter and T-NOS terminators, selective marker genes of NPT II, HPT, Bar, and aadA, and GUS reporter gene. Overall, qualitative and quantitative PCR tests infer that the constructed plasmid is a suitable and costeffective strategy for the detection and quantification of GM tobacco.

Data Availability
All the data generated or analyzed during this study are included in the main paper and supplementary information files.

Ethical Approval
Not applicable.

Consent
Not applicable.

Conflicts of Interest
e authors declare that there are no conflicts of interest regarding the publication of this paper.