CN112760413A - Application of public primer-mediated multiple quantitative PCR detection technology in transgenic soybean detection - Google Patents
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Abstract
The invention discloses application of a public primer-mediated multiple quantitative PCR detection technology in transgenic soybean detection, in particular to detection of 6 types of transgenic soybeans which are allowed to be imported by rural parts in agriculture in China. 5 'to each pair of specific primers',A section of common primer is added at both ends to form a specific chimeric primer. Specific fragments are amplified by the multiple PCR detection system, the length of each transgenic soybean fragment is detected by GeXP, the detection sensitivity of the GeXP multiple detection system can reach 1 ng/mu L, and the amplification product sequencing result further shows the specificity of primer amplification. The multiple PCR quantitative detection technology based on the GeXP system can accurately identify 6 transgenic soybeans, and provides a novel detection method for the transgenic soybeans and other transgenic products.
Description
Technical Field
The invention belongs to a transgenic soybean detection technology, and particularly relates to a research on a public primer-mediated multiple quantitative PCR detection technology and an application thereof in transgenic soybean detection.
Background
The safety problem of transgenic food is always a hotspot problem of society, and the establishment of a high-throughput detection method capable of identifying the transgenic food has important significance for the right of knowledge of consumers. The nucleic acid detection is the main technology of transgenic food detection and the inevitable trend of future development, and the food nucleic acid detection technology mainly comprises PCR series technology (such as single PCR, multiple PCR, fluorescent quantitative PCR and the like), digital PCR technology, Southern hybridization, constant temperature amplification technology, gene chip technology, high-throughput sequencing and the like. Because the detected food ingredients are not clear, the current food nucleic acid detection technology mainly adopts multiplex PCR, fluorescent quantitative PCR, gene chip and high-throughput sequencing technology. The fluorescent quantitative PCR technology is limited by a fluorescent detection channel, and the detection target is generally within 6. The gene chip technology and the high-throughput sequencing are mainly applied to gene diagnosis of human bodies, can be technically applied to food detection, but the food detection is low in charge, and the technology cannot meet the economic and rapid detection requirements of food. At present, multiple PCR has the advantages of high efficiency, sensitivity, economy, simplicity and the like because multiple target molecules are simultaneously detected in one PCR tube, and is widely concerned in the food safety nucleic acid detection technology. However, the technology is mainly used for qualitative analysis at present and is difficult to carry out quantitative detection.
Disclosure of Invention
The invention combines multiple PCR amplification with GeXP high-throughput detection, introduces a common primer with fluorescent labeling on the basis of the traditional PCR, combines the common primer and the specific primer to synthesize a new strand after the specific primer is combined with a template, and uses the new strand as a new template strand for amplification. After several cycles, the amplified products are all fluorescently labeled. The Size of bp of the product can be obtained through the DNA Standard Size provided by the GeXP system, qualitative analysis is carried out on a detection sample, the relative content of the detection sample can be determined and quantitative analysis can be realized through detecting the fluorescence signal intensity of the product through the GeXP, for example, a Standard curve is established, and the research on the aspect is relatively less at present.
The invention adopts the following technical scheme:
the method comprises the steps of extracting genomic DNA of a product to be detected as a template, carrying out PCR reaction by adopting a two-step annealing temperature method in the presence of an embedded primer and a fluorescent common primer, and carrying out GeXP system detection on a PCR product to finish the detection of transgenic soybean components in the product.
The invention discloses a method for detecting transgenic soybean components in a product by utilizing a public primer-mediated multiple quantitative PCR detection technology, which comprises the following steps of extracting genomic DNA of the product to be detected as a template, carrying out PCR reaction by adopting a two-step annealing temperature method in the presence of a chimeric primer and a fluorescent public primer, and carrying out GeXP system detection on the PCR product to finish the detection of the transgenic soybean components in the product.
The invention discloses application of a chimeric primer and a fluorescent public primer in multiple quantitative PCR detection of transgenic soybean components in a product.
In the invention, the sequence of the common primer is SEQ ID NO.1, and Cy5 fluorescent dye labeling is carried out at the 5' end of the common primer; the chimeric primers are SEQ ID NO.2 to SEQ ID NO. 13.
In the present invention, the varieties of transgenic soybean are MON7701, DP356043, CV-127, MON87769, MON87705 and MON 87708.
In the invention, when the PCR reaction is carried out, the temperature rise program is 95 ℃ for pre-denaturation for 5 min; denaturation at 95 ℃ for 30s, annealing at 62 ℃ for 10s, annealing at 56 ℃ for 20s, and extension at 72 ℃ for 30s for 10 cycles; then carrying out pre-denaturation at 72 ℃ for 2 min; denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 30s for 30 cycles; finally, extension was carried out at 72 ℃ for 7 min. The first 10 cycles, creatively designing the target sequence amplified by the specific primer in the chimeric primer with low concentration at two annealing temperatures, and generating a PCR product with a public primer end; in the last 30 cycles, the high concentration of the common primer was amplified in large amounts using the PCR product with the common primer end generated earlier as a template at a low annealing temperature (corresponding to the second annealing temperature in the previous cycle). The method can effectively and simply detect whether the products, especially the food contain the transgenic soybean ingredients, and makes great contribution to national right of knowledge and food safety. A25 microliter system was used for the PCR reaction, with a final concentration of 1600nM of common primers and the following final concentration of chimeric primer pairs for transgenic soybeans: m7SF53.6nM, M7SR 53.6.6 nM, D6SF 52.8.8 nM, D6SR 52.8nM, CVSF 24nM, CVSR 24nM, M69SF16nM, M69SR 64nM, M05SR 40nM, M08SF32 nM; the product is food, feed, etc. containing soybean component.
According to a public primer mediated multiple technology established by earlier research, fluorescence is marked at the 5' end of a public primer, fluorescent quantitative analysis is carried out on different product fragments by utilizing the characteristic that all extension products have the public primer, a nucleic acid quantitative detection technology with high flux, high accuracy and reliability suitable for food detection is developed, and a quantitative detection method is established for 6 transgenic soybeans imported in China, including MON7701, DP356043, CV-127, MON87769, MON87705 and MON87708, so that a new detection means is provided for market supervision of the transgenic soybeans.
Drawings
FIG. 1 shows the results of specificity and sensitivity assays for transgenic soybean MON87701, in which template concentrations of A, B, C, D and E were 20, 2, 1, 0.2, and 0.02 ng/. mu.L, respectively, and the lowest assay template concentration was 1 ng/. mu.L.
FIG. 2 shows the results of specificity and sensitivity tests of transgenic soybean DP356043, wherein the concentrations of A, B, C, D and E are 20, 2, 1, 0.2 and 0.02 ng/. mu.L, respectively, and the lowest test template concentration is 1 ng/. mu.L.
FIG. 3 shows the results of the specificity and sensitivity test of transgenic soybean CV-127, in which the template concentrations of A, B, C, D and E were 20, 2, 1, 0.2 and 0.02 ng/. mu.L, respectively, and the lowest test template concentration was 0.2 ng/. mu.L.
FIG. 4 is a graph showing the results of specificity and sensitivity assays for transgenic soybean MON87769, wherein the template concentrations of A, B, C, D and E were 20, 2, 1, 0.2, and 0.02 ng/. mu.L, respectively, and the lowest assay template concentration was 0.2 ng/. mu.L.
FIG. 5 is a graph of the specificity and sensitivity assay of transgenic soybean MON87705, where the template concentrations of A, B, C, D and E were 20, 2, 1, 0.2, 0.02 ng/. mu.L, respectively, and the lowest assay template concentration was 1 ng/. mu.L.
FIG. 6 is a graph of the specificity and sensitivity assay of transgenic soybean MON87708, wherein the template concentrations of A, B, C, D and E were 20, 2, 1, 0.2, 0.02 ng/. mu.L, respectively, and the lowest assay template concentration was 1 ng/. mu.L.
FIG. 7 shows the results of sequencing the amplification products.
Detailed Description
T30DPCR apparatus, LG2020 gel imaging analysis system, CK-6 normal temperature high speed centrifuge (Hangzhou Langzhou scientific instruments, Inc.), BXM-30R autoclave (Shanghai Bochn medical bio-instruments, Inc.), MICROCL17 centrifuge, Nanodrop 2000/2000C spectrophotometer (Sammer Feishale science, Inc.), ‒ 80 deg.C low temperature refrigerator (Sanyo electric appliances, Inc., Japan), MSX (SD EE) electronic analytical balance (Meitler-Tokia, Switzerland), DDY-5 model Voltage and Current stabilized electrophoresis apparatus (Beijing Hexayi instruments, Inc.), DK-600B electrothermal constant temperature sink (Shanghai Jinghai Macro Corp.), GNP-9050 water-isolated constant temperature incubator (Shanghai Jinghai instruments, Inc.), BCD-118 refrigerator (Heifei Mei Milli, Inc.), Air tech clean bench (Sujing Corp 5810R low temperature high speed centrifuge, Inc.), Micropipettes (Eppendorf, Germany), GeXP analysis system (Beckman, USA).
Transgenic soybean MON87701, CV127, MON87769, MON87708, MON87705 powder standards (American society for oil and fat chemistry), transgenic soybean DP356043 (European Union of Standard), GeXP start Kit, DNA size standard Kit-400 Base Pairs, isolation buffer and isolation gel (Beckman, USA), centrifugal column type plant genome DNA extraction Kit (Beijing Tiangen corporation), common primer (Suzhou jin Weizhi corporation), PCR premix 2 XDreamAMTAqGreen, DNA molecular weight standard Ladder (Fermas corporation, USA), agar powder (Beijing Kangcheng Cheng Hai Biotech Co., Ltd.), gel stain (Beijing Quanyun Co., Ltd.), chloroform, phenol, absolute ethanol (analytical purity, pharmaceutical Co., Ltd.), DNA Dye (6 XDDNA Load Dye, Thermo corporation), fluorescent public primer (Cy 5 is labeled at 5' end, suzhou jinviru corporation). The transgenic soybean insertion gene, characteristics and national standard detection method are shown in table 1.
Examples
Transgenic soybean (MON87701, DP356043, MON87769, CV-127, MON87705, MON87708) genomes were extracted according to the instructions provided by the plant genome DNA extraction kit of Beijing Tiangen corporation, and the extracted DNA was stored in a refrigerator at ‒ 20 ℃.
The primers are shown in Table 2.
Note that the downstream primers of MON87708 and MON87769 are the same, and the upstream primers of MON87701 and MON87705 are the same; the 5' end of CP4 was labeled with Cy5 fluorescent dye.
In the UP-MPCR (common primer-mediated multiplex PCR) system of this example, the sequences of the internal reference primers are GGGTGAGGATAGGGTTCTCTG at the upstream and GCGATCGAGTAGTGAGAGTCG at the downstream, the product length is 210 bp, and the reaction of the multiplex system is not affected.
The basic working principle of traditional PCR is to amplify a DNA target fragment by using a DNA molecule to be amplified as a template and simultaneously using a pair of oligonucleotide fragments which are respectively complementary with the template as primers for amplification. The common primer mediated multiple PCR introduces the common primer based on the traditional PCR. After the specific primer is connected to the template strand, the common primer is connected with the specific primer, so that a new DNA strand containing the common primer is generated, the chimeric primer mainly serves as a template for subsequent common primer amplification, the subsequent amplification can be performed by using the common primer for PCR amplification, the concentration of the chimeric primer can be greatly reduced by the amplification principle, the interaction among multiple primers is effectively reduced, and the multiple PCR detection flux is improved.
mu.L of 2 XTaq master Mix, 4. mu.L of fluorescent public primer, 2. mu.L of chimeric primer (mixture), 1. mu.L of DNA template, 5.5. mu.L of dH2And (3) performing amplification by using a multiplex PCR amplification system of O. And taking the amplified sample as a detection object of GeXP. The details of the CP-MPCR amplification system and the program are as follows, 2 XTaq master Mix 12.5. mu.L, fluorescent common primer (10. mu. mol/L) 4. mu.L, chimeric primer (mixture) 2. mu.L, DNA template 1. mu.L, and finally distilled water to 25. mu.L. Wherein the composition of the chimeric primer mixture comprises 6.7 mu L R: 6.7 muL of M7F, 6.6 mu L R: 6.6 muL of D6F, 3 mu L R: 3 muL of CV F, 1 mu L R: 8 muL of M69F, 4 muL of M05R and 4 muL of M08F. Then make up to 100. mu.L with distilled water. The initial DNA template concentration was 20 ng/. mu.L. The final concentration of the fluorescent common primer is 1.6 mmol/L, and the final concentrations of systems of the chimeric primer pairs are M7 SF/R53.6 nmol/L, D6 SF/R52.8 nmol/L, CVSF/R24 nmol/L, M69SF16 nmol/L, M69SR 64 nmol/L, M05SR 40 nmol/L and M08SF32 nmol/L respectively. Among these primers, the downstream primers of MON87708 and MON87769 are the same, and MON87701 is the same as the upstream primer of MON 87705.
The CP-MPCR amplification procedure was 95 deg.C (5 min), 95 deg.C (30 s), 62 deg.C (10 s), 56 deg.C (20 s), 72 deg.C (30 s), 72 deg.C (2 min), 95 deg.C (30 s), 56 deg.C (30 s), 72 deg.C (7 min). Wherein, the 2 nd to the 4 th steps are 10 cycles, and the 7 th to the 9 th steps are 30 cycles.
Specific detection of GeXP multiplex PCR:
first, a mixture of SDS and DSS (DNA Standard size) was prepared by adding 80-fold volume of SLS to DSS400, and vortexing and mixing. And then, adding 40 muL of mixed liquor into each sample hole, adding 0.1-1 muL of PCR product, checking whether bubbles exist, and adding one drop of paraffin oil. Then, 300 μ L of separation buffer was added to the corresponding buffer dish wells. Finally, the separation was carried out using the method of Frag 3 (name of method, generally 6 kV, 40 min). After the detection of the instrument is finished, each hole has a corresponding peak image, and the corresponding bp (basepair) number can be read according to the result of DSS shown on the peak image.
When the concentration of the used DNA template is 20 ng/. mu.L, the measured product length is shown in figure 1-figure 6, the MON87701 is 140.75 bp, the DP356043 is 184.42 bp, the CV-127 is 274.93 bp, the MON87769 is 308.47 bp, the MON87705 is 467.98 bp, the MON87708 is 517.67 bp, and the minimum amplification fragment of the detection system is 140 bp, which indicates that the method has good specificity.
Sensitivity detection of GeXP multiplex PCR:
mu.L of 2 XTaq master Mix, 4. mu.L of fluorescence-labeled common primer, 2. mu.L of chimeric primer (mixture), 1. mu.L of DNA template, 5.5. mu.L of dH2O, wherein the concentration of the DNA template is diluted from 20 ng/. mu.L to 2, 1, 0.2 and 0.02 ng/. mu.L respectively. And respectively amplifying the 5 concentrations, then, putting the amplification products into a GeXP system for detection, and judging GeXP multiplex PCR sensitivity according to whether a peak exists in the finally obtained image. The concentration of the DNA template is respectively diluted to 5 different concentrations of 20 ng/muL, 2 ng/muL, 1 ng/muL, the DNA template is amplified through multiplex PCR, the amplified product is placed into a GeXP system for detection, the result is shown in figures 1-6, the amplified band size and the lowest concentration capable of being detected of each transgenic soybean are shown in a table 3, the sensitivity of 2 transgenic soybeans of CV-127 and MON87769 reaches 0.2 ng/muL, the sensitivity of the rest 4 transgenic soybeans reaches 1 ng/muL, and the detection sensitivity can be detected by the transgenic soybeans.
Note that the bp number is originally an integer, and the bp numbers provided in the above table are calculated by the GeXP system according to the marker condition, so that decimal numbers appear.
Sequencing of amplification products:
in order to further confirm the specificity of the amplification product, the detection results of the GeXP systems in fig. 1 to 6 show that the sizes of all detection fragments are consistent with the target amplification fragment, in order to further explain that the amplification fragments are the detection fragments, the PCR reaction product is sent to soviet jinweizhi company for sequencing, fig. 7 is a partial sequencing result diagram, and the sequencing result shows that the sequences of the amplification fragments are consistent with the corresponding sequences of all transgenes.
Comparative example
The primer sequence disclosed by the invention is the same as CN 2018105173895, 6 gene templates are selected according to the primers in CN 2018105173895 table 1 to carry out CP-MPCR amplification and GeXP system detection in the same way as the embodiment, the result is found to be changed, which shows that the selection of the primers has influence on the detection performance, and the specific steps are as follows:
the creativity of the invention is not in the design of primers, but in the selection of 6 primers and the adoption of a GeXP method after amplification, so that the detection limit of partial transgenic products can reach 0.2 ng/mu L. The invention adopts the common primer-mediated multiple PCR technology, and the common primer has no homologous sequence in the genome of the detection target, so the influence of the multiple PCR primers and the common primer on the annealing temperature is small. However, the common primer is added to the 5' end of each pair of multiplex primers as a consensus sequence, so that the multiplex primers are generally over 40 bp, and the design of the multiplex primers mainly needs to consider whether the multiplex primers added with the common primer have secondary structures, whether dimers form among the primers, the binding efficiency between the primers and the template, and the like. Compared with the fluorescent quantitative PCR technology, the nucleic acid detection technology can realize the quantitative detection of a plurality of amplification products only by marking one fluorescence on the common primer, not only breaks through the detection flux limitation caused by the fluorescent detection channel of the fluorescent quantitative PCR, but also has the advantages of simpler method and lower detection cost because the adopted common primer marks single fluorescence. By adopting the research scheme, 6 transgenic soybean varieties of MON87701, DP356043, CV-127, MON87769, MON87705 and MON87708 are used as experimental objects, and the specificity and the sensitivity of the GeXP to the transgenic soybean varieties are detected respectively. The result shows that when the concentration of the DNA template of the system is 20 ng/. mu.L, M7 has a characteristic peak at 140.75 bp, D6 has a characteristic peak at 184.42 bp, CV has a characteristic peak at 274.93 bp, M05 has a characteristic peak at 467.98 bp, and M08 has a characteristic peak at 517.67 bp, the sequencing result shows that target sequences are amplified, and the 6 transgenic soybeans can be specifically identified by using the system. When the concentration of the DNA template in the system is diluted to 2, 1, 0.2 and 0.02 ng/. mu.L, CV and M69 can still detect characteristic peaks at the concentration of 0.2 ng/. mu.L, and M7, D6, M05 and M08 can detect the characteristic peaks at the lowest concentration of 1 ng/. mu.L, so that the system has higher sensitivity for identifying the several transgenic soybeans. Compared with the common PCR which can only observe the result through gel electrophoresis, GeXP provides a basis for further quantitative analysis research.
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Claims (10)
1. The application of a public primer-mediated multiple quantitative PCR detection technology in transgenic soybean detection is characterized in that genomic DNA of a product to be detected is extracted as a template, a two-step annealing temperature method is adopted for PCR reaction in the presence of a chimeric primer and a fluorescent public primer, and GeXP system detection is carried out on a PCR product to complete detection of transgenic soybean components in the product; the sequence of the public primer is SEQ ID NO.1, and the 5' end of the public primer is marked by fluorescent dye.
2. The application of the public primer-mediated multiple quantitative PCR detection technology in the detection of transgenic soybeans according to claim 1, wherein the chimeric primer is SEQ ID No.2 to SEQ ID No. 13.
3. The application of the public primer-mediated multiple quantitative PCR detection technology in the detection of the transgenic soybeans according to claim 1, wherein in the PCR reaction, the temperature rise program is 95 ℃ for 5 min; denaturation at 95 ℃ for 30s, annealing at 62 ℃ for 10s, annealing at 56 ℃ for 20s, and extension at 72 ℃ for 30s for 10 cycles; then carrying out pre-denaturation at 72 ℃ for 2 min; denaturation at 95 ℃ for 30s, annealing at 56 ℃ for 30s, and extension at 72 ℃ for 30s for 30 cycles; finally, extension was carried out at 72 ℃ for 7 min.
4. The use of the common primer-mediated multiplex quantitative PCR detection technology in the detection of transgenic soybean according to claim 1, wherein a 25 microliter system is used in the PCR reaction, wherein the final concentration of the common primer is 1600nM, and the final concentration of the chimeric primer pair for transgenic soybean is as follows: m7SF53.6nM, M7SR 53.6.6 nM, D6SF 52.8.8 nM, D6SR 52.8.8 nM, CVSF 24nM, CVSR 24nM, M69SF16nM, M69SR 64nM, M05SR 40nM, M08SF32 nM.
5. The use of the public primer-mediated multiplex quantitative PCR detection technology in the detection of transgenic soybeans according to claim 1, wherein the product is food or feed, health care product or medicine containing soybean components.
6. The application of the chimeric primer and the fluorescent public primer in the transgenic soybean component in the multiplex quantitative PCR detection product; the sequence of the public primer is SEQ ID NO.1, and the 5' end of the public primer is marked by fluorescent dye.
7. The use of claim 6, wherein the chimeric primer is SEQ ID No.2 to SEQ ID No. 13.
8. The use of claim 6, wherein the varieties of transgenic soybean are MON7701, DP356043, CV-127, MON87769, MON87705, and MON 87708.
9. The method for detecting the transgenic soybean components in the product by using the public primer-mediated multiple quantitative PCR detection technology is characterized by comprising the following steps of extracting the genomic DNA of the product to be detected as a template, carrying out PCR reaction by adopting a two-step annealing temperature method in the presence of a chimeric primer and a fluorescent public primer, and carrying out GeXP system detection on the PCR product to finish the detection of the transgenic soybean components in the product.
10. The kit for detecting the transgenic soybean by the multiplex quantitative PCR comprises a chimeric primer and a fluorescent common primer; the sequence of the common primer is SEQ ID NO.1, and fluorescent dye labeling is carried out at the 5' end of the common primer; the chimeric primers are SEQ ID NO.2 to SEQ ID NO. 13.
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| CN114807406A (en) * | 2022-03-07 | 2022-07-29 | 江汉大学 | Primer pair combination, kit and detection method for detecting soybean transgenic components |
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