CN114369676A - Primer combination, kit, detection method and application for detecting tobacco transgenic components - Google Patents

Primer combination, kit, detection method and application for detecting tobacco transgenic components Download PDF

Info

Publication number
CN114369676A
CN114369676A CN202210001378.8A CN202210001378A CN114369676A CN 114369676 A CN114369676 A CN 114369676A CN 202210001378 A CN202210001378 A CN 202210001378A CN 114369676 A CN114369676 A CN 114369676A
Authority
CN
China
Prior art keywords
tobacco
transgenic
detection
primer
detecting
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Granted
Application number
CN202210001378.8A
Other languages
Chinese (zh)
Other versions
CN114369676B (en
Inventor
李甜甜
李论
彭海
周俊飞
肖华锋
万人静
陈利红
高利芬
方治伟
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Jianghan University
Original Assignee
Jianghan University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Jianghan University filed Critical Jianghan University
Priority to CN202210001378.8A priority Critical patent/CN114369676B/en
Publication of CN114369676A publication Critical patent/CN114369676A/en
Application granted granted Critical
Publication of CN114369676B publication Critical patent/CN114369676B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
    • C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/13—Plant traits
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/16—Primer sets for multiplex assays
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/166—Oligonucleotides used as internal standards, controls or normalisation probes

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Engineering & Computer Science (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Health & Medical Sciences (AREA)
  • Analytical Chemistry (AREA)
  • Biotechnology (AREA)
  • Molecular Biology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Immunology (AREA)
  • Biophysics (AREA)
  • Physics & Mathematics (AREA)
  • Genetics & Genomics (AREA)
  • Biochemistry (AREA)
  • Microbiology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Botany (AREA)
  • Mycology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

The invention relates to the technical field of biology, in particular to a primer combination, a kit, a detection method and application for detecting tobacco transgenic components. The primer pair combination for detecting the tobacco transgenic components comprises primer pairs for amplifying 15 common detection transgenic elements and 1 tobacco internal reference gene sequence NT _ UBI. The invention also relates to a kit and a detection method for detecting the tobacco transgenic components. The technical scheme of the invention is simple to operate and high in detection efficiency; the detection objects are comprehensive, and the method has the characteristics of high detection specificity, accuracy, sensitivity and the like; can be used for large-scale detection of transgenic tobacco and products thereof, and has better application prospect.

Description

Primer combination, kit, detection method and application for detecting tobacco transgenic components
Technical Field
The invention relates to the technical field of biology, in particular to a primer combination, a kit, a detection method and application for detecting tobacco transgenic components.
Background
Tobacco (Nicotiana tabacum L.) belongs to the solanaceae family, the genus Nicotiana. Originating from oceania, america, and the southern pacific, some islands. Tobacco is an important economic crop and the first sharp industry of all countries in the world, and the planting area is very large. How to improve the quality and yield of tobacco and stress resistance is a concern of researchers. The problem is ameliorated by the transgenic technology to obtain tobacco plants and their progeny, since tobacco is readily tissue cultured and transformed. However, the safety problem of transgenic products has been regarded as a problem. Therefore, the development of efficient and convenient transgenic detection technology is very important.
The detection technology of transgenic products mainly comprises a protein-based detection method and a nucleic acid-based detection method. At present, the PCR detection method based on nucleic acid is still the most common and accurate transgene detection technology at present, and mainly comprises methods such as common qualitative PCR, nested PCR, loop-mediated isothermal amplification (LAMP), fluorescent quantitative PCR multiplex PCR and the like. Compared with the common qualitative PCR method, the nested PCR method has high detection sensitivity and is easy to cause false positive. LAMP is simple to operate and strong in specificity, however, primer design is complex, DNA pollution is easily caused, and subsequent experiments are affected. The fluorescent quantitative PCR method has the advantages of good repeatability, high sensitivity and less nucleic acid cross-contamination, but has high cost and needs special detection instruments. The common multiplex PCR method can detect a plurality of genes in one reaction at the same time, but generally the detection is not more than six times, otherwise, the interference between primers is large, and the detection effect is influenced. The gene chip and the digital PCR technology are also common transgenic product detection technologies, have the advantages of high flux, high sensitivity, strong specificity and the like, and can detect a plurality of genes in 1 transgenic crop in parallel or simultaneously detect a plurality of transgenic crops; however, the cost is high, special instruments and equipment are needed, and operators are required to have high professional quality, and the factors limit the wide application of the technology in detection.
Therefore, the development of a high-efficiency, sensitive and high-flux transgenic product detection method becomes a key problem to be solved urgently.
Disclosure of Invention
In order to solve the technical problems, the general idea of the embodiment of the application is as follows:
the nucleotide sequence of the detection element of the tobacco transgenic product, namely the target molecule and the reference gene are screened to be used as the detection target. The method comprises 15 detection common transgenic elements: p35S, t35S, pFMV35S, tNOS, PAT, bar, NPtII, Hpt, CP4epsps, E93, TMV-CP, pVY _ CP, CMV _ CP, Cry1Ab and GUS and the sequence NT _ UBI comprising the tobacco internal reference gene 1.
Next, the present invention developed a multiplex PCR primer composition for detecting said transgenic elements and tobacco reference genes, wherein 15 pairs were directed to 15 transgenic elements and 1 pair was directed to 1 reference gene. The primers do not conflict with each other, and efficient amplification can be performed by multiplex PCR. The multiplex PCR primer composition can be used for developing a transgenic element detection kit.
The specific technical scheme is that in the first aspect, the application provides a primer pair combination for detecting tobacco transgenic components, the primer pair combination comprises 15 primers numbered as NtGMO1, NtGMO2, NtGMO3, NtGMO4, NtGMO5, NtGMO6, NtGMO7, NtGMO8, NtGMO9, NtGMO10, NtGMO11, NtGMO12, NtGMO13, NtGMO14 and NtGMO15, each primer pair consists of a forward primer and a reverse primer, and the nucleotide sequence of the specific primer pair combination is shown as SEQ ID NO.1-SEQ ID NO. 30.
Also provides a pair of primer pair combination with the number of NtGMO16 for amplifying the tobacco internal reference gene NT _ UBI, and the nucleotide sequence is shown as SEQ ID NO.31-SEQ ID NO. 32.
These primers were used to amplify the following tobacco transgenic elements, respectively: p35S, t35S, pFMV35S, tNOS, PAT, bar, NPtII, Hpt, CP4epsps, E93, TMV-CP, pVY _ CP, CMV _ CP, Cry1Ab and GUS, wherein the nucleotide sequences of the primers and the tobacco transgenic elements amplified by the primers, namely the target molecules, are specifically corresponding to the numbers of the corresponding primer pairs and the nucleotide sequences of the primers are shown in Table 1.
TABLE 1 target molecules screened according to the invention and their primer sequences
Figure BDA0003454385440000021
When the primers are designed, in order to enhance the applicability and sensitivity of the primers, the length of the primers is between 18 and 30bp, the primers are not interfered with each other, and all the primers can be combined into a primer pool for multiplex PCR amplification, namely all the designed primers can be normally amplified in one amplification reaction.
In another aspect, the present application provides a kit for detecting tobacco transgenic components, wherein the kit comprises the primer pair combination for detecting tobacco transgenic components of claim 1 and the primer pair combination for amplifying tobacco internal reference gene NT _ UBI of claim 2.
Preferably, the detection kit further comprises a multiplex PCR master mix.
The primer pair combination of claim 1 or 2 and the detection kit of claim 3 or 4 are also provided for detecting transgenic tobacco seeds and related products.
Also provided is a method for detecting the transgenic components of tobacco, which is characterized by comprising the following steps:
1) carrying out reference on a tobacco transgenic element and a tobacco reference gene to obtain a multiple PCR primer;
2) obtaining the DNA of the tobacco to be detected; adding the multiple PCR primers into a reaction system by taking the DNA as a template to perform amplification reaction to obtain an amplification product; performing high-throughput sequencing on the amplification product to obtain a high-throughput library; and analyzing the gene sequences in the high-throughput library to realize the detection of tobacco transgenic components.
Preferably, the method comprises an amplification reaction environment/program comprising: pre-denaturation at 94 ℃ for 15 min; the first step of amplification reaction, denaturation at 94 ℃ for 20 seconds, annealing at 65-57 ℃ and extension for 60 seconds, 10 Touch Down cycles, (the temperature of annealing and extension in each cycle is reduced by 0.8 ℃); the second amplification reaction, denaturation at 94 ℃ for 20 seconds, annealing at 57 ℃ and extension for 60 seconds, 26 cycles.
Still preferably, the reaction system of the process comprises: total 30 μ l, primer pair: 2. mu.l, 2 XBuffer: 15ul, multiplex amplification enzyme: 0.5 mul; supplementing the rest water; the high throughput library concentration of greater than 2ng/ul is acceptable.
In order to realize the purpose of detecting tobacco transgenic components in a sample, when the tobacco transgenic element is selected, a detection primer for a tobacco internal reference gene is added, and the content of the transgenic components is quantitatively detected.
When the primers are designed, in order to enhance the applicability and sensitivity of the primers, the length of the primers is between 18 and 30bp, the primers are not interfered with each other, and all the primers can be combined into a primer pool for multiplex PCR amplification, namely all the designed primers can be normally amplified in one amplification reaction.
Specifically, when the components of the multiplex PCR premix comprise the combination of the transgenic element of the tobacco and the primers of the internal reference gene, each primer is premixed according to the proportion of 1:1, the primers are mixed according to different experimental purposes, and in the specific implementation example, the concentration of each primer is 2 nM.
In some embodiments, the primer pair ranges are: the number of pairs 1 to 16 is appropriately adjusted depending on the condition of a specific test sample. In the later period, the number of the newly collected transgenic elements can be increased periodically, and 3000 pairs of primer combinations are tried, so that the amplification effect is still good. To achieve the detection of transgenic components in tobacco, we collected 16 pairs of commonly used tobacco transgenic elements, and the log range of the multiplex PCR primers was: 1-16 pairs, compared with the conventional 8-pair specific multiplex PCR, has the advantages of high detection flux and high sensitivity.
In particular, high throughput sequencing can be second generation sequencing or third generation sequencing, and the resulting high throughput library can analyze the components of the transgene from multiple dimensions, including but not limited to the transgene elements in our embodiment.
In some embodiments, the method can be used for detecting all target transgenic components of multiple samples at one time, has the advantages of high throughput, high sensitivity, accuracy, rapidness and the like, and can be applied to qualitative and quantitative detection of transgenic components of tobacco and tobacco products thereof.
The kit provided by the invention can sensitively detect the transgenic components with the content of 0.05% in the sample.
In the reproducibility test of the invention, the detection result reproducibility r of each sample among different libraries and different library establishing batches is 100%, and the accuracy rate a is 100%.
The kit provided by the invention has high specificity in detecting various transgenic components in a complex template.
The invention has the beneficial effects that:
1) the method is simple to operate, multiple samples or multiple transgenic components in one sample can be synchronously detected through one-time sample pretreatment, single-tube PCR amplification, library construction and sequencing, the characteristics of parallel analysis and multiple judgment are realized, and the detection efficiency of transgenic products is greatly improved;
2) the detection objects are complete and comprise the current common transgenic element sequence and transgenic strain of tobacco, and a new detection sequence can be conveniently added, so that the amplification failure of a single target is avoided, and the specificity, accuracy and sensitivity of detection are improved;
3) the kit is fused with a second-generation sequencing platform to sequence the amplified product, so that the detection flux and the repeatability of the system are improved, and the detection result can be directly digitalized and is suitable for large-scale detection of the transgenic tobacco and the products thereof. Therefore, the invention overcomes the defects of time and labor waste and high cost in the prior art, and the provided tobacco transgenic detection kit has the advantages of simple operation, rapidness, sensitivity, large detection flux, good repeatability of detection results and low detection cost of multi-sample multi-target sequences, and has important application to the detection of transgenic products at seed stations, agricultural institutions and customs entry and exit ports.
The technical scheme of the application will be described in detail by combining examples, comparative examples and experimental data.
Drawings
FIG. 1; structural schematic diagram of transgenic tobacco
Detailed Description
In order to facilitate an understanding of the invention, the invention is described in more detail below with reference to the accompanying drawings and specific examples. Preferred embodiments of the present invention are shown in the drawings. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.
It is to be noted that, unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.
Unless otherwise specifically stated, various raw materials, reagents, instruments, equipment and the like used in the examples of the present invention are commercially available or can be prepared by an existing method.
Example 1 screening of target transgene Components and design of multiplex PCR amplification primers
S1 screening of target transgenic component
In the embodiment of the application, the target transgenic components, namely, the transgenic elements and the reference genes, are comprehensively collected from a common transgenic database, a national standard, an industrial standard or the existing literature as far as possible so as to ensure the specificity and the accuracy of detection. Wherein the names of the selected transgenic elements and the reference gene are shown in the above table 1:
s2 design of multiplex PCR amplification primers
In the embodiment of the application, Primer3Plus is used for designing multiple PCR primers, the length of the primers is between 18-30bp, the primers are not interfered with each other, the dimer between the primers or the hairpin structure in the primers and the non-specific amplification of non-target sequences are mainly evaluated, all the primers after evaluation can be combined into a Primer pool for multiple PCR amplification, namely all the designed primers can be normally amplified in one amplification reaction. Specific primer sequences include: SEQ ID NO.1-SEQ ID NO. 32.
Example 2 detection of tobacco samples containing transgenic Components
1. Experimental materials: transgenic material of over-expressed tobacco OE-GUS-7 and OE-CBF1-3 was stored in this experiment.
Preparation of DNA template: the extraction of plant genome adopts a high-efficiency plant genome DNA extraction kit (DP350) of CTAB or Tiangen Biochemical technology (Beijing) limited company. In this example, DNA of a sample to be tested was extracted using a Tiangen DNA extraction kit, and each sample was subjected to three biological replicates.
PCR amplification, library construction and sequencing
Amplifying the genome DNA of the sample by using 30 pairs of multiplex PCR amplification primers; connecting the amplification product of each sample with a sequencing adaptor and a specific sample DNA bar code, and mixing to obtain a high-throughput sequencing library; and detecting the high-throughput sequencing library by using a high-throughput sequencing platform and performing quality control on the high-throughput sequencing data. In the step, key parameters such as amplification cycle number and sequencing depth need to be researched and adjusted according to requirements such as detection accuracy and sensitivity; the step can also be connected with the step of the third-generation sequencing related topic so as to realize the advantage complementation between the second-generation sequencing and the third-generation sequencing.
4. Determination of the results
1) Determining whether contamination is acceptable according to the signal index S of the transgenic component in the test sample and the signal index P of the transgenic component in the blank, wherein: the blank noise index P ═ Nc/Nc, where Nc and Nc represent the number of sequenced fragments of transgenic components and the total number of sequenced fragments in the blank, respectively. The signal index S of the test sample is Nt/Nt, where, and Nt represent the number of sequencing fragments of the transgenic component and the total number of sequencing fragments in the test sample, respectively. S/P ratio
2) Determination of transgenic results
And distributing each sequencing fragment to each target position of each target species by using the DNA bar code of the sample to be tested and the homologous alignment, wherein the target comprises a transgenic element and an internal reference gene. Absolute quantification of the transgene components was achieved based on the number of sequenced sequences at each target position. When the sequence sequences on the internal reference gene and the transgenic element are compared to exceed a specified threshold, qualitatively judging that the sample contains transgenic components; when the sample contains the transgenic component, the content of the exogenous gene in the sample is quantitatively judged according to the ratio of the sequencing sequence of the transgenic element and the reference gene.
The calculation formula of the transgene content in this example is shown in (A):
Figure BDA0003454385440000061
ctest DNA-transgene content of test sample
tTi-number of sequencing sequences per transgenic element in a test sample
tRi-number of sequencing sequences of each reference Gene fragment detected in test sample
m-total number of reference Gene fragments detected in test sample
n-total number of detected transgenic element fragments in the Standard
According to the present example, we tested a total of 2 samples, each of which was replicated in three organisms, and the results are shown in Table 2 and FIG. 1: promoters and terminators commonly used in negative samples will also detect several sequences in negative tobacco species, i.e., in this example, require that sequences with less than 5 reads be filtered out. The present invention provides that contamination in the detection system can be judged to be acceptable when the signal-to-noise ratio is greater than 10 times. When the signal-to-noise ratio of the transgene component in the sample is greater than 10, the nucleic acid of the transgene component in the sample is determined to be detected. Specifically, all transgenic elements in the OE-GUS-7 sample were effectively detected in three replicates and were approximately 10% in content; from this table it is shown that this tobacco transgene kit of our invention can be used to detect transgene products.
TABLE 2 transgenic test results of the test samples of this example 2
Figure BDA0003454385440000062
Figure BDA0003454385440000071
Example 3 evaluation of accuracy, specificity and sensitivity
Transgenic standards of herbicide-resistant tobacco varieties OE-GUS-7 and OE-CBF1-3 were prepared at different mass percentages to evaluate the accuracy and sensitivity of the developed technology. Specifically, the transgene content of each sample is diluted by mass percent, specifically, transgenic tobacco OE-GUS-7 and OE-CBF1-3 are respectively diluted into 10%, 1%, 0.1%, 0.05%, 0.025% and 0.01% samples by negative tobacco, and the diluted sample numbers respectively correspond to the diluted sample numbers of transgenic lines RT-73 (A1, A2, A3, A4, A5, A6) and the diluted sample numbers of OE-CBF1-3 (B1, B2, B3, B4, B5 and B6). The accuracy of qualitative detection refers to the proportion of true positives to true negatives, and the quantitative accuracy refers to the degree of coincidence between the average of multiple determinations and the true value, expressed as an error. The specificity is also called true negative rate, and the true negative detected by multiple detections accounts for the percentage of all negatives. Sensitivity refers to the lowest level of transgene content that can be detected at 95% confidence, i.e., the lower limit of detection. The assay was performed according to the method of example 2, with three biological replicates per sample, and the results are shown in table 3: the kit can stably detect each transgenic element in a sample with the transgenic content of 0.05 percent, can detect at most 1 transgenic component in a negative sample, has strong specificity, can obviously distinguish the sample with the transgenic content of 0.05 percent from the negative sample, and has technical stability and detection sensitivity with the transgenic content of 0.05 percent.
TABLE 3 evaluation of the accuracy and sensitivity of the method of the invention
Figure BDA0003454385440000072
Figure BDA0003454385440000081
Note: + represents detected, -represents not detected, a1 and B1 represent 10% transgene content, a2 and B2 represent 1% transgene content, A3 and B3 represent 0.1% transgene content, a4 and B4 represent 0.05% transgene content, a5 and B5 represent 0.025% transgene content, and a6 and B6 represent 0.01% transgene content.
Example 4 application of the method of our invention to the actual detection of samples
In order to verify the accuracy of the invention and the function of the batch sample in transgenic detection, 108 tobacco leaf samples of unknown genotypes of a certain company are selected in a laboratory for detection, the detection is carried out according to the method of the embodiment 2, the detection result is compared with the storage type of the company, and the consistency of the result is counted. Analysis results show that only 2 samples among 108 test samples have inconsistent results, and the consistency of the detection results is up to 98.1%, so that the accuracy and the good application prospect of the method are better proved.
It is noted that, in this document, relational terms such as "first" and "second," and the like, may be used solely to distinguish one entity or action from another entity or action without necessarily requiring or implying any actual such relationship or order between such entities or actions. Also, the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article, or apparatus. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other identical elements in a process, method, article, or apparatus that comprises the element.
Sequence listing
<110> university of Jianghan
<120> primer combination, kit, detection method and application for detecting tobacco transgenic components
<130> 20220102
<160> 32
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 1
cgcgtacaca acaagtcagc 20
<210> 2
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 2
tgtcgtcacc ttcaccttcg 20
<210> 3
<211> 21
<212> DNA
<213> Artificial sequence ()
<400> 3
agggtttcgc tcatgtgttg a 21
<210> 4
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 4
tcggtacccc tggattttgg 20
<210> 5
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 5
gatgcttttg gtaggtgcgc 20
<210> 6
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 6
tccagcacat gcatcatggt 20
<210> 7
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 7
ttgccggtct tgcgatgatt 20
<210> 8
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 8
aacatagatg acaccgcgcg 20
<210> 9
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 9
gtctatggag gcgcaaggtt 20
<210> 10
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 10
catgccatcc accatgcttg 20
<210> 11
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 11
gacaagcacg gtcaacttcc 20
<210> 12
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 12
gagacgtaca cggtcgactc 20
<210> 13
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 13
caagatggat tgcacgcagg 20
<210> 14
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 14
tcagagcagc cgattgtctg 20
<210> 15
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 15
attcccaata cgaggtcgcc 20
<210> 16
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 16
atttgtgtac gcccgacagt 20
<210> 17
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 17
aaatcctctg gcctttccgg 20
<210> 18
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 18
ccttccttac ggatcctggc 20
<210> 19
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 19
ttcattgcgc acacaccaga 20
<210> 20
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 20
agaggccacg atttgacaca 20
<210> 21
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 21
gtggccataa ggagcgctat 20
<210> 22
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 22
caagttgcag gaccagaggt 20
<210> 23
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 23
aaatctgcgg gatgtgggtt 20
<210> 24
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 24
tggtgtgcct ctctgtgttc 20
<210> 25
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 25
cagtatgctg catccggagt 20
<210> 26
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 26
agatgtggga atgcgttggt 20
<210> 27
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 27
tggaaactac accgaccacg 20
<210> 28
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 28
ataccttggg cagaaccacg 20
<210> 29
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 29
ggtgcacggg aatatttcgc 20
<210> 30
<211> 20
<212> DNA
<213> Artificial sequence ()
<400> 30
ataacggttc aggcacagca 20
<210> 31
<211> 19
<212> DNA
<213> Artificial sequence ()
<400> 31
tccaggacaa ggagggtat 19
<210> 32
<211> 21
<212> DNA
<213> Artificial sequence ()
<400> 32
catcaacaac aggcaaccta g 21

Claims (8)

1. A primer pair combination for detecting tobacco transgenic components is characterized by comprising 15 pairs of primers which are numbered NtGMO1, NtGMO2, NtGMO3, NtGMO4, NtGMO5, NtGMO6, NtGMO7, NtGMO8, NtGMO9, NtGMO10, NtGMO11, NtGMO12, NtGMO13, NtGMO14 and NtGMO15, wherein each pair of primers consists of a forward primer and a reverse primer, and the nucleotide sequence of the specific primer pair combination is shown as SEQ ID NO.1-SEQ ID NO. 30.
2. The primer pair combination according to claim 1, further comprising a pair of primers with the nucleotide sequence shown in SEQ ID NO.31-SEQ ID NO.32, wherein the primer pair is numbered NtGMO16 and is used for amplifying the tobacco internal reference gene NT _ UBI.
3. A kit for detecting tobacco transgenic components, which is characterized by comprising the primer pair combination for detecting tobacco transgenic components in claim 1 and the primer pair combination for amplifying tobacco internal reference gene NT _ UBI in claim 2.
4. The test kit of claim 3, further comprising a multiplex PCR premix.
5. The primer pair combination of claim 1 or 2, and the detection kit of claim 3 or 4, are used for detecting transgenic tobacco seeds and related products.
6. A method for detecting a transgenic component of tobacco, comprising the steps of:
1) carrying out reference on a tobacco transgenic element and a tobacco reference gene to obtain a multiple PCR primer;
2) obtaining the DNA of the tobacco to be detected; adding the multiple PCR primers into a reaction system by taking the DNA as a template to carry out amplification reaction to obtain an amplification product; performing high-throughput sequencing on the amplification product to obtain a high-throughput library; and analyzing the gene sequences in the high-throughput library to realize the detection of the tobacco transgenic components.
7. The method of claim 6, wherein the environment/program of the amplification reaction comprises: pre-denaturation at 94 ℃ for 15 min; the first step of amplification reaction, denaturation at 94 ℃ for 20 seconds, annealing at 65-57 ℃ and extension for 60 seconds, 10 Touch Down cycles, (the temperature of annealing and extension in each cycle is reduced by 0.8 ℃); the second amplification reaction, denaturation at 94 ℃ for 20 seconds, annealing at 57 ℃ and extension for 60 seconds, 26 cycles.
8. The method of claim 7, wherein the reaction system comprises: total 30 μ l, primer pair: 2. mu.l, 2 XBuffer: 15ul, multiplex amplification enzyme: 0.5 mul; supplementing the rest water; the high-throughput library was found to be at a concentration of greater than 2 ng/ul.
CN202210001378.8A 2022-01-04 2022-01-04 Primer combination, kit, detection method and application for detecting transgenic components of tobacco Active CN114369676B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210001378.8A CN114369676B (en) 2022-01-04 2022-01-04 Primer combination, kit, detection method and application for detecting transgenic components of tobacco

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210001378.8A CN114369676B (en) 2022-01-04 2022-01-04 Primer combination, kit, detection method and application for detecting transgenic components of tobacco

Publications (2)

Publication Number Publication Date
CN114369676A true CN114369676A (en) 2022-04-19
CN114369676B CN114369676B (en) 2024-03-22

Family

ID=81141494

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210001378.8A Active CN114369676B (en) 2022-01-04 2022-01-04 Primer combination, kit, detection method and application for detecting transgenic components of tobacco

Country Status (1)

Country Link
CN (1) CN114369676B (en)

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1718743A (en) * 2005-07-04 2006-01-11 黑龙江省烟草科学研究所 Transgene tobacco detecting method and reagent box
CN105567830A (en) * 2016-01-29 2016-05-11 江汉大学 Method for detecting transgenic ingredients of plant
CN109988858A (en) * 2017-12-29 2019-07-09 贵州中烟工业有限责任公司 A kind of transgene tobacco multiple fluorescence PCR gene loci, primer and its detection method
US20190241981A1 (en) * 2016-02-01 2019-08-08 Fraunhofer-Gesellschaft Zur Foerderung Der Angewandten Forschung E.V. Plant breeding using next generation sequencing
CN113512608A (en) * 2021-06-11 2021-10-19 贵州中烟工业有限责任公司 LAMP (Loop-mediated isothermal amplification) detection primer system, kit and method for transgenic tobacco

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN1718743A (en) * 2005-07-04 2006-01-11 黑龙江省烟草科学研究所 Transgene tobacco detecting method and reagent box
CN105567830A (en) * 2016-01-29 2016-05-11 江汉大学 Method for detecting transgenic ingredients of plant
US20190241981A1 (en) * 2016-02-01 2019-08-08 Fraunhofer-Gesellschaft Zur Foerderung Der Angewandten Forschung E.V. Plant breeding using next generation sequencing
CN109988858A (en) * 2017-12-29 2019-07-09 贵州中烟工业有限责任公司 A kind of transgene tobacco multiple fluorescence PCR gene loci, primer and its detection method
CN113512608A (en) * 2021-06-11 2021-10-19 贵州中烟工业有限责任公司 LAMP (Loop-mediated isothermal amplification) detection primer system, kit and method for transgenic tobacco

Also Published As

Publication number Publication date
CN114369676B (en) 2024-03-22

Similar Documents

Publication Publication Date Title
CN114107552B (en) Primer pair combination, kit, detection method and application for detecting rape transgenic line
CN114507749A (en) Primer group, kit and method for accurately detecting corn transgenic components
CN114622028B (en) Primer pair combination, kit and detection method for detecting transgenic papaya
CN106521018A (en) Primer and method for high-flux detection of transgenic maize containing NOS terminator
CN109161609B (en) SNP molecular marker of wheat leaf rust resistance gene Lr42, detection method and application
CN111378781A (en) Molecular marker primer for quickly and efficiently identifying salt-tolerant gene SKC1 of rice and application
CN114369676B (en) Primer combination, kit, detection method and application for detecting transgenic components of tobacco
CN114369678B (en) Primer combination, kit, detection method and application for detecting transgenic components and transgenic lines of beet
CN114369675B (en) Primer combination, kit, detection method and application for detecting transgenic components and transgenic lines of potatoes
CN114196782B (en) Primer combination, kit, detection method and application for detecting transgenic components and transgenic lines of tomatoes
CN114277178B (en) Primer pair combination for detecting transgenic components of rape, kit, detection method and application
CN114807406B (en) Primer pair combination, kit and detection method for detecting soybean transgenic component
CN114807407B (en) Primer pair combination, kit and detection method for detecting soybean transgenic strain
CN114774566B (en) Primer pair combination, kit and detection method for detecting cotton transgenic component
CN114606335A (en) Development and application of KASP molecular marker of sugarcane mosaic virus disease resistance gene of corn
CN114369677A (en) Primer combination, kit, detection method and application for detecting wheat transgenic components and transgenic strains
CN114657275B (en) Primer pair combination, kit and detection method for detecting transgenic alfalfa
CN114774567B (en) Primer pair combination, kit and detection method for detecting transgenic components of rice
CN114657276B (en) Primer pair combination, kit and detection method for detecting rice transgenic line
CN115491428A (en) Primer pair combination, kit and detection method for detecting cotton transgenic line
CN116121437B (en) SNP (single nucleotide polymorphism) marker combination of mangiferin fruit variety and application of SNP marker combination in mangiferin fruit breeding
CN114507750B (en) Primer group, kit and detection method for detecting corn transgenic line
CN116334290B (en) Primer group and kit for identifying rice functional genes and application of primer group and kit
CN117535387A (en) Rice core SNP marker combination developed based on liquid phase chip and KASP technology and application thereof
CN114032322A (en) Development and application of SNP molecular marker of maize flowering phase gene

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant