Applicability of plasmid calibrant pMON87712 for quantitative detection of the transgenic soybean MON87712

Abstract

The transgenic glyphosate-tolerant soybean MON87712 event was developed by the agrochemical and agricultural biotechnology company Monsanto (USA) and commercialized in 2013. Due to the absence of matrix-based and genomic DNA-positive reference material for MON87712, it is very difficult to detect and monitor this event. In this study, we developed a recombinant 760-bp linearized plasmid, including 150 bp of the soybean endogenous lectin gene and 610 bp of the exogenous BBX32 gene plus its 3ʹ flanking sequence of MON87712 by In-Fusion cloning technology. In addition, a duplex real-time polymerase chain reaction for the detection of MON87712 and the soybean endogenous lectin gene was established. By using this method, we achieved specific and quantitative detection of MON87712 in 45 other kinds of crops, with a detection limit of 10 copies/μl. This method provides a new technical means for the accurate detection of transgenic soybean MON87712, as well as technical support for the supervision of agricultural transgenic organisms.

Introduction

As the main source of oil crops and plant-based protein, soybean is an important industrial raw material and occupies an important position in China’s food security and national economy [1]. Since the development of the world’s first transgenic soybean with tolerance to glyphosate [2], there have been continuous advances in the cultivation of new varieties of transgenic crops, playing an evident role in improving crop production and also showing great potential. The safety of transgenic crops has always been the focus of public concern; therefore, an objective method for the evaluation and detection with a higher safety evaluation standard has become the basis for the safe management of transgenic crops. Since the establishment of accurate detection methods is a prerequisite to promoting this aim further, accurate, reliable, and widely applicable quantitative assays are urgently required to provide the corresponding technical support.

MON87712 was developed by the agrochemical and agricultural biotechnology company Monsanto (USA). Transgenic traits were introduced into plants through Agrobacterium tumefaciens–mediated transformation. The transgenic traits included tolerance to glyphosate and enhanced photosynthesis/yield. In 2013, MON87712 was approved for planting in the USA. However, until 2020, the USA was the only country that approved MON87712 for use as a feed or raw material [3]. Few studies of MON87712 have been reported [4], and no reference materials (RMs) are available.

RMs play an important role in the development and evaluation of new methods, calibration of instruments, and routine analysis in many fields [5]. In genetically modified organism (GMO) analysis, matrix-based RMs, genomic DNA RMs, and plasmid DNA RMs are the mainly used RMs. Currently, commercial matrix-based and genomic DNA-certified reference materials are mainly produced by the Reference Materials and Measurements, European Commission, and the American Oil Chemists’ Society (AOCS). In 2002, Kuribara et al. [6] developed plasmid reference molecules for genetically modified (GM) soybean and maize quantification through a collaborative ring trial. Since then, more and more plasmid reference molecules have begun to be used as calibrators in GMO detection [7–9]. The main advantages of plasmid reference molecules are that they can be easily constructed and produced with high quantity and quality, multiple GM sequences from different GM events can be recombined into one plasmid, and one plasmid can be used as calibrator for multiple GM events [10]. Plasmid reference molecules are now widely accepted in GMO analysis [11–15]. However, the matrix-based and genomic DNA-positive RMs for MON87712 are still needed for traceability requirements.

In this study, we developed a recombinant 760-bp linearized plasmid consisting of 150 bp of the soybean endogenous lectin gene and 610 bp of the exogenous BBX32 gene plus its 3ʹ border flanking sequence that is event specific for MON87712 (the junction sequence). This junction sequence is the nucleotide sequence that spans the point at which heterologous DNA inserted into the genome is linked to the soybean cell genomic DNA, and the detection of this sequence in a biological sample–containing soybean DNA is diagnostic of the presence of the soybean event MON87712. In addition, we proposed a duplex real-time polymerase chain reaction (PCR) method for the detection of MON87712 and the soybean endogenous lectin gene. Using this method, we achieved specific and quantitative detection of MON87712.

Materials and Methods

Materials, primers, and probes

The 46 test materials used in this study are shown in Table 1. The primers and probes for event-specific sequence of MON87712 were designed using Primer Premier 5.0 software (PREMIER Biosoft International, San Francisco, USA). The primers and probes for the soybean endogenous lectin gene were adopted from the Chinese National Standard for GMO detection [16]. The sequence of the soybean endogenous lectin gene was obtained from GenBank (accession No. K00821.1), and the sequence of MON87712 was obtained from a patent document [17]. The probe for the 5′ end of the soybean endogenous lectin gene was fluorescently labeled with hexachloro-6-methylfluorescein (HEX) (TaKaRa, Dalian, China). The 5′ end of the probe for the event-specific MON87712 gene was labeled with 6-carboxyfluorescein (FAM) (TaKaRa). The sequences of the primers and probes and the fluorescent labels used in this study are shown in Table 2. The primers and probes were synthesized by TaKaRa.

Genomic DNA extraction

The plant product samples used in this study were ground into uniform powder with a mixer (34BL99; Waring Blender Dynamics Corp., New Hartford, USA). DNA was extracted by using the Mini BEST Plant Genomic DNA Extraction Kit (TaKaRa). After the extracted genomic DNA was dissolved in 100 µl 10× Tris-EDTA (TE; pH 7.6) buffer (Qingdao JISSKANG Technology Co., Ltd, Qingdao, China), the nucleic acid concentration was determined by using the Nanodrop ND-1000 ultra-micro spectrophotometer (ThermoFisher, Wilmington, USA). DNA samples were stored at –20°C for later use.

Preparation of linearized plasmid

Due to the lack of a commercial MON87712 RM, it is necessary to generate plasmids by artificial synthesis. According to the sequences of the soybean endogenous lectin gene and the MON87712 event [17], 150 bp of the lectin gene and 610 bp of the exogenous BBX32 gene and its 3ʹ border flanking sequence inserted into MON87712 were synthesized (TaKaRa) artificially.

In-Fusion cloning (TaKaRa) technology relies on the homologous sequences at the end of vector and insertion fragment to complete seamless cloning. The 15-bp sequence at the end of the linearized vector pMD-19T (TaKaRa) was added to the 5′ end of the PCR primer of the inserted fragment. The fragment with the complementary ends for insertion into the vector was obtained by PCR. The primer sequences used in In-Fusion clone are as follows: IFF, 5′-GATCCTCTAGAGATTCATCCACATTTGGGACAA-3′, and IFR, 5′-TGCAGGTCGACGATTAAAAGATAAGTTTATCAC-3ʹ (TaKaRa). The reaction system contained the following: 10 μl 5× PrimeSTAR buffer (Mg²⁺ Plus), 4 μl dNTP mixture (2.5 mM each), 10 pmol primer IFF, 10 pmol primer IFR, 100 ng template, 0.5 μl PrimeSTAR HS DNA Polymerase (2.5 U/μl) (TaKaRa), and sterilized dH₂O added to give a final volume of 50 μl. The reaction conditions were as follows: 98°C 10 s, 55°C 5 s, 72°C 1 min/kb, 30 cycles.

The In-Fusion enzyme, linear vector, and PCR products were mixed in the following reaction system: 2 μl 5× In-Fusion HD Enzyme Premix (TaKaRa), 1 μl linearized vector, 1 μl purified PCR fragment, and dH₂O added to give a final volume of 10 μl. The reaction system was incubated at 50°C for 15 min and then placed on ice. The whole amount (10 μl) was added into 100 μl JM109 competent cells and cultured overnight on an LB-agar plate containing 40 μg/ml X-gal (Invitrogen, Carlsbad, USA), 24 μg/ml isopropyl-beta-D-thiogalactoside (IPTG) (Invitrogen), and 100 μg/ml ampicillin (Invitrogen).

The length of the inserted fragment was confirmed by PCR. Screening was performed with the universal primers of pMD19-T vector. Reaction system: 1 μl plasmid supernatant, 10 μl 2× Premix Ex Taq (TaKaRa), 5 μM pMD19-T vector universal primers Tvector-1/Tvector-2 (1 μl each), and dH₂O were added to give a final volume of 25 μl. Reaction parameters: pre-denaturation at 95°C for 4 min, denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 30 s; 30 cycles.

According to the results of PCR screening, positive clones were further cultured and the plasmids were extracted for sequencing by TaKaRa. Previous studies [18] have shown that the amplification efficiency of closed-loop target sequence DNA is lower than that of linear DNA; therefore, plasmid samples should be linearized for use as a template. The plasmids verified by sequencing were linearized by XbaI digestion as follows: 1.5 μl XbaI (15 U/μl), 5 μl 10× M buffer, 5 μl 0.1% bovine serum albumin, 5 μl pMON87712, and dH₂O were added to give a final volume of 50 μl. The reaction was incubated at 37°C for 3 h. The concentration of the linearized plasmid pMON87712 was 77.1 ng/μl.

Reaction system of a duplex real-time PCR for MON87712

The total reaction system of 25 µl contained 12.5 µl 2× Premix Ex Taq (Probe qPCR), 1 μl 10 μM forward primer MON87712-F1, 1 μl 10 μM reverse primer MON87712-R1, 1 μl 5 μM probe MON87712-P1, 1 μl 10 μM forward primer Lec-F, 1 μl 10 μM reverse primer Lec-R, 1 μl 5 μM probe Lec-P, and 2 μl linearized plasmid pMON87712. The parameters of the duplex real-time PCR were as follows: pre-denaturation at 95°C for 30 s followed by 40 cycles of 95°C for 5 s, 55°C for 10 s, and 72°C for 20 s. Generally, cycle threshold (Ct) values ≥40 were classified as negative, while Ct values ≤35 were accepted as positive. When Ct values were in the range between 35∼40, the template concentration adjustment was required and a repetition of the real-time fluorescent PCR was done, and if the Ct values were still in the range between 35∼40, we assumed it to be positive.

Reproducibility, sensitivity, and quantification limit

Duplex real-time PCRs for the 46 test materials were performed to verify the specificity of the primers and probes for MON87712 and lectin gene. The test concentration of the linearized plasmid pMON87712 was 100 copies/μl. The conversion between DNA sample and copy number [16] was calculated as follows:

where cp_DNA is the copies of DNA, cont_DNA is the DNA amount in nanograms, and len_DNA is genomic DNA length (bp). According to this equation, the number of copies of linearized plasmid pMON87712 was calculated, and serial dilutions (5×10⁶, 5×10⁵, 5×10⁴, 5×10³, 5×10², 50, 25, 12.5, 5, 0.5, and 0.05 copies/μl) were prepared in TE buffer. According to the experimental requirements, different concentrations were selected to test the reproducibility, sensitivity, and quantification limit. Six replicates were tested for each concentration. The relative standard deviation (RSD) analysis of the Ct value was used to analyze the duplex real-time PCR results.

Establishment of standard curves

The standard curves for the soybean endogenous lectin gene and the event-specific sequence of MON87712 were established according to the formula: |${Ct} = K \times \lg A + B$|⁠, where Ct is the number of cycles in which the fluorescence signal exceeded the corresponding threshold in the real-time PCR, K is the slope of the curve, A is the number of copies, and B is the intercept. The duplex real-time PCR was performed using gradient dilutions of MON87712 linearized plasmid (5×10⁶, 5×10⁵, 5×10⁴, 5×10³, 5×10², 50, and 5 copies/μl) as a template. Six replicates were tested for each concentration.

Homogeneity and stability of plasmid calibrant pMON87712

For the homogeneity test, 15 tubes were randomly selected from the plasmid calibrant pMON87712 samples. Each sample was tested three times, and the sampling amount was 2 μl/reaction. The samples were tested by duplex real-time PCR, and the Ct values were evaluated for homogeneity using the analysis of variance method.

For the stability test, the plasmid calibrant pMON87712 samples were subject to prolonged storage at –18°C to –20°C. Three samples were randomly selected for analysis on Days 0, 46, 91, and 136, and each sample was repeated three times. The samples were tested by duplex real-time PCR, and the Ct values were evaluated for stability using the analysis of variance method.

Results and Discussion

Linearized plasmid

The linearized plasmid generated had a total length of 760 bp, and the nucleotide sequence data reported are available in the GenBank databases (submission number 2386841). The proposed primers and probes sites for the event-specific sequence of MON87712 and the soybean endogenous lectin gene in linearized plasmid are shown in Fig. 1.

Primer specificity

The soybean endogenous lectin gene and event-specific sequence of MON87712 amplification products were separated by 2% agarose gel electrophoresis (Fig. 2). The fragments amplified using the primer pairs Lec-F/Lec-R and MON87712-F1/MON87712-R1 were 74 and 107 bp in length, respectively. The results indicated successful amplification by the primers and probes specific for the event-specific sequence of MON87712 by using pMON87712 as a template, while no amplification was observed (Table 3) using the other 45 test materials, such as soybean (except MON87712), corn, rape, alfalfa, red bean, mung bean, peanut, rice, GM cottonseed, barley, and wheat. Similarly, successful amplification was achieved by the primers and probes specific for the soybean endogenous lectin gene using pMON87712 and another 18 types of soybean as templates (Table 3), while no amplification was observed using the 27 test materials such as corn, rape, alfalfa, red bean, mung bean, peanut, rice, GM cottonseed, barley, and wheat (Table 3). These results demonstrated good specificity of the tested primers and probes. The duplex real-time PCR amplification profile of MON87712 linearized plasmid is shown in Fig. 3.

Standard curve establishment

In accordance with the regulations of the European Union Reference Laboratories (EURL) relating to the various parameters of the quantitative real-time PCR (qPCR) standard regression curve [19], the linear correlation coefficient of the standard curve R²≥0.98 and amplification efficiency E [Efficiency[%]= (10 (–1/slope)–1)×100%] were in the range of 90%–110%, and the slope ranged from –3.1 to –3.6. In this study, the standard curve equation of the soybean endogenous lectin gene was Y = –3.093 lgX+39.77, the standard curve equation of the event-specific sequence of MON87712 was Y = –3.196 lgX+39.81, and each parameter in the qPCR standard curve equation was within the specified range. The qPCR amplification and standard curves of the soybean endogenous lectin gene are shown in Fig. 4, and the qPCR amplification and standard curves of the event-specific sequence of MON87712 are shown in Fig. 5. These results confirmed that the method is suitable for specific and quantitative analysis of MON87712.

Reproducibility analysis

The reproducibility of a PCR is an important indicator that affects the quantitative analysis of transgenes. Real-time PCR detection was performed on serial dilutions of the linearized plasmids pMON87712 (10⁵, 10⁴, 10³, 10², 50, 25, and 10 copies/μl) (Table 4). For the Ct value of the soybean endogenous lectin gene, the SD range was 0.04~0.29, and the RSD range was 0.16%–0.82%. For the Ct value of the event-specific sequence of MON87712, the SD range was 0.13–0.90, and the RSD range was 0.40%–2.65%. All these parameters complied with the threshold range for the RSD (≤25%) stipulated by EURL. These data indicated that the reproducibility of the method is acceptable.

Detection sensitivity and quantification limit

Duplex real-time PCR detection sensitivity was analyzed using nine concentrations of MON87712 linearized plasmids (10⁵, 10⁴, 10³, 10², 50, 25, 10, 1, and 0.1 copies/μl) (Table 5). Successful detection was achieved in the concentration range from 10⁵ to 0.1 copies/μl, suggesting that the sensitivity of this method is acceptable, with a detection limit of 0.1 copies/μl. However, at concentrations of 1 and 0.1 copies/μl, the RSD of the six replicates exceeded the threshold range of 25%, which showed that the precision of the detection at these concentrationμs was poor. In contrast, the precision of the quantitative analysis of samples containing >10 copies/μl was less than the acceptance criterion of 25%. Hence, the lower limit of detection of this method is 10 copies/μl.

Homogeneity and stability analysis

The samples were tested by duplex real-time PCR, and the Ct values were evaluated for homogeneity using the analysis of variance method. The estimated standard deviation of quality (SD) was 0.045. When α=0.05, f1=14, and f2=30, the F critical value was 2.04, and the F ratio was 1.55, which is less than the F critical value, indicating that there is no significant difference in and between the tubes, and the sample homogeneity meets the requirements (Table 6). The SD was 0.030. When α=0.05, f1=11, and f2=24, the F critical value was 2.22, and the F ratio of the Ct value was 1.86, which is less than the F critical value, indicating that the properties of the tested samples are stable over time (Table 7).

Conclusion

In summary, a novel reference plasmid pMON87712, which targets GM soybean MON87712 event-specific sequence, as well as the endogenous lectin gene was constructed. This is the first report of the use of plasmid for the detection of MON87712. With advances in genetical modification technology and increased commercialization, the GM components in imported products or commercially available soybean products are becoming more and more complex. In the actual testing process, it is common that samples are mixed, with low purity, or integration of multiple copies. In this study, we developed a duplex real-time PCR of GM soybean events and analyzed its specificity and sensitivity and detection range. The results demonstrated that the proposed primers and probes for MON87712 only produced fluorescent signals relating to target soybean events in duplex real-time PCR, indicating the specificity of the amplification. Hence, this method is suitable for the screening and identification of GM soybeans, as well as the accurate detection of the content of GM components in soybeans, with a quantitative detection limit reaching 10 copies/μl. The establishment of this method not only provides strong technical support for GM regulation but also adds a new technical resource for the future management of GM product labeling thresholds.

Funding

This work was supported by the grant from the Major Project for Cultivation of GMO New Varieties of China (No. 2018ZX08012-001).

Conflict of Interest

The authors declare that they have no conflict of interest.

References