CN113718018A - Method for analyzing wheat gene copy number by optimized digital PCR method - Google Patents
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Abstract
The invention discloses a method for analyzing the copy number of a wheat gene by adopting an optimized digital PCR method, which comprises the following steps: 1) extracting the genomic DNA of the wheat to be detected; 2) carrying out enzyme digestion treatment on the genomic DNA extracted in the step 1) by using restriction enzyme; 3) carrying out microdroplet digital PCR reaction; 4) and after the PCR reaction is finished, determining the number of positive droplets and negative droplets of each hole of the sample of the PCR reaction plate, and obtaining the gene copy number of the wheat to be detected. The digital PCR template of the invention has low requirement, and compared with the sensitivity of real-time PCR of 0.1 pg.mu.L, the sensitivity of the digital PCR reaches 0.001 pg.mu.L, which is more than 100 times of the real-time PCR. Compared with Southern, the digital PCR method has short test period, simple operation and high detection flux.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a method for analyzing the copy number of a wheat gene by adopting an optimized digital PCR method.
Background
Droplet-based digital PCR (ddPCR) is a nucleic acid detection technique that has recently emerged. The technology does not depend on any calibrator, and realizes absolute quantification of nucleic acid detection by adopting the principle of directly counting single molecules. ddPCR has been widely used in many fields of life science research, such as absolute quantification of nucleic acids, rare mutation detection, and copy number detection. The ddPCR has the characteristic of high accuracy, and when the copy number of a target gene is detected, the copy number of the target gene can be quickly obtained by utilizing the double reaction of the target gene and a reference gene and calculating the ratio of the target gene and the reference gene. This method has become the first choice for detecting gene copy number due to the good reproducibility of experimental results. Compared with the traditional method, ddPCR has obvious advantages, mainly reflects in that the operation process is simpler, a standard curve is not required to be constructed, the absolute quantification is directly carried out on the nucleic acid molecules to be detected, and the test result is more accurate and reliable. In addition, ddPCR is superior to qRT-PCR in linear range, detection limit, quantification limit, etc. Research shows that ddPCR has been used in detecting transgene insert copy number of corn, rice and other crops. However, due to the huge and complicated genome (about 17Gb) of hexaploid wheat, the copy number detection by using the traditional methods such as Southern blot and the like is time-consuming and labor-consuming, and cannot meet the requirement of high-throughput detection, and when the copy number analysis is required to be performed on a large number of transformation events, the traditional methods cannot meet the requirement, so that it is very important to develop a high-throughput and accurate copy number analysis method. However, copy number analysis methods based on digital PCR methods for wheat are still lacking at present.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: a method of copy number analysis based on a digital PCR method for wheat is provided. The established high-throughput and accurate transgenic wheat copy number analysis method can be used for large-scale screening of transgenic wheat transformation events, and the screening efficiency of excellent and stable transformation events is improved.
The invention provides a digital PCR detection method for detecting the copy number of a wheat gene, which comprises the following steps:
1) extracting the genomic DNA of the wheat to be detected;
2) carrying out enzyme digestion treatment on the genomic DNA extracted in the step 1) by using restriction enzyme;
3) carrying out microdroplet digital PCR reaction;
4) and after the PCR reaction is finished, determining the number of positive droplets and negative droplets of each hole of the sample of the PCR reaction plate, and obtaining the gene copy number of the wheat to be detected.
Wherein, the wheat genome DNA to be detected is extracted by combining a CTAB method and a full-automatic nucleic acid extractor in the step 1).
Wherein, in the step 2), the restriction enzyme is EcoRI.
Wherein the enzyme digestion treatment method in the step 2) comprises the steps of treating overnight at 37 ℃, treating for 30min at 65 ℃ to inactivate enzyme, and adding 1 unit of EcoRI into each microgram of wheat genome DNA as the dosage of the restriction enzyme.
Wherein, the PCR reaction system in the step 3) is as follows: 2 XddPCRSuperMix 10. mu.l, 500nmol/L of target gene forward primer 1. mu.L, 500nmol/L of target gene reverse primer 1. mu.L, 500nmol/L of reference gene forward primer 1. mu.L, 500nmol/L of reference gene reverse primer 1. mu.L, 250nmol/L of target gene probe 0.5. mu.l, 250nmol/L of reference gene probe 0.5. mu.l, wheat genome DNA template 5. mu.l, total volume 20. mu.l.
Wherein the concentration of the wheat genome DNA template is 20 ng/. mu.L.
Wherein the forward primer of the target gene is Nib8-5-F, and the Nib8-5-F is a single-stranded DNA molecule shown in a sequence 1; the reverse primer of the target gene is Nib8-5-R, and the Nib8-5-R is a single-stranded DNA molecule shown in a sequence 2; the target gene probe is Nib8-5-P, and the Nib8-5-P is a single-stranded DNA molecule shown in a sequence 3; the reference gene forward primer is PINb-D1b-F, and the PINb-D1b-F is a single-stranded DNA molecule shown as a sequence 4; the reference gene reverse primer is PINb-D1b-R, and the PINb-D1b-R is a single-stranded DNA molecule shown in a sequence 5; the reference gene probe is PINb-D1b-P, and the PINb-D1b-P is a single-stranded DNA molecule shown in a sequence 6.
The 5 'end and the 3' end of the target gene probe Nib8-5-P are respectively connected with a HEX label and a BHQ1 label; the reference gene probe is formed by connecting a FAM label and a BHQ1 label to the 5 'end and the 3' end of PINb-D1b-P respectively.
Wherein, the PCR reaction conditions in the step 3) are as follows: at 95 ℃ for 10 min; at 94 ℃, 3s, 59 ℃, 1min, for 40 cycles; at 98 deg.C for 10 min.
The invention has the beneficial effects that: the digital PCR template of the invention has low requirement, and compared with the sensitivity of real-time PCR of 0.1 pg.mu.L, the sensitivity of the digital PCR reaches 0.001 pg.mu.L, which is more than 100 times of the real-time PCR. Compared with Southern, the digital PCR method has short test period, simple operation and high detection flux. The invention can establish a high-throughput and accurate transgenic wheat copy number analysis method, can be used for large-scale screening of transgenic wheat transformation events, and improves the screening efficiency of excellent and stable transformation events.
Drawings
FIG. 1 shows the results of digital PCR experiments at different annealing temperatures for the nib8 gene and the reference gene.
FIG. 2 shows the results of digital PCR experiments with different template concentrations for nib8 gene and reference gene.
FIG. 3 shows the results of the nib8 strain-specific test microdroplet.
FIG. 4 shows southern results of nib8 transgenic wheat 16 plants.
Detailed Description
The present invention is described in further detail below with reference to specific embodiments, which are given for the purpose of illustration only and are not intended to limit the scope of the invention. The examples provided below serve as a guide for further modifications by a person skilled in the art and do not constitute a limitation of the invention in any way.
The experimental procedures in the following examples are conventional unless otherwise specified. Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Example 1
Construction of digital PCR detection system
1.1 test materials
Transformed nib8 gene (Durable field resistance to white yellow mosaic virus in transgenic wheat mutant fusion gene [ J ]. Plant Biotechnology Journal,2014,12(4):447 456.) and wheat Yangma 16 (recipient).
1.2 primer design of reference Gene and target Gene
A single copy homozygous gene PINb-D1 (grain hardness) located in a wheat D genome is selected as an internal reference gene for a test, primers with strong specificity and specificity are determined through screening and comparison of the primers, and the specificity of the probe is ensured (Table 1).
TABLE 1 digital PCR Probe primer sequences
1.3 detection method
1.3.1 extraction of sample DNA:
the genomic DNA of the wheat leaves to be detected is extracted by combining a CTAB method with a full-automatic nucleic acid extractor extraction method (Wangguan, Square-Honn. plant genetic engineering, second edition, Beijing: scientific Press, 2002).
1.3.2 fragmented genomes
The leaf genomic DNA was subjected to endonuclease pretreatment, and the genome was digested with EcoRI from NEB corporation in an experiment in which 1 unit of EcoRI was added per microgram of DNA. The enzyme was inactivated by treatment at 37 ℃ overnight and at 65 ℃ for 30 min.
The template DNA was diluted to 20. mu.g/. mu.L, and PCR test was carried out using QX200DDPCR system, a product of Bio-Rad, Inc., of Beijing agricultural Biotechnology research center.
1.3.3 preparation reaction System and PCR
PCR is performed by a probe method using DNA as a template. The reaction systems and PCR procedure are shown in tables 2 and 3.
TABLE 2 PCR amplification reaction System
TABLE 3 PCR amplification reaction procedure
Generation of oil droplets in the PCR system: adding 20 mu L of PCR reaction system and 70 mu L of microdroplet generation oil (droplet generation oil) into a special 8-channel microdroplet generation clamping groove (droplet generator DG8 cartridge), covering a matched rubber pad (droplet generator DG8 gasket), putting into a microdroplet generator to generate microdroplets, generating the microdroplets in the uppermost row of the microdroplet generation card, wherein the volume of the microdroplets is 40 mu L, transferring the microdroplets into holes at corresponding positions of a 96-well plate, and sealing the microdroplets by using a preheated PX1 heat sealing instrument.
Microdroplet PCR: and carrying out amplification on a PCR instrument, wherein the temperature rise and fall speed of PCR is required to be lower than 2 ℃/s in order to ensure efficient template amplification.
1.3.4 droplet analysis
The 96-well plate is placed in a droplet analyzer and the fluorescence signal of the droplet is read, thereby judging the positivity/negativity of the droplet. The number of microdroplets (copies/. mu.L) of the target sequence of each sample was calculated using QuantaSoft V1.3.2 software, and then the number of copies of the target gene to be tested was calculated from the number of copies of the internal reference gene.
Example 2
Digital PCR assay at different annealing temperatures
According to the method of example 1, the annealing temperatures in step 1.3 are changed from 57 ℃ to 56 ℃ (A01), 56.9 ℃ (B01), 58.1 ℃ (C01), 59 ℃ (D01), 59.6 ℃ (E01), 60 ℃ (F01), and the numbers in brackets are used to reflect the results of the digital PCR test, and the digital PCR test results are shown in FIG. 1, the results of the digital PCR test at different annealing temperatures of the nib8 gene and the internal reference gene in FIG. 1, the A diagram is the results of the nib8 gene, and the B diagram is the internal reference gene. Blue dots are positive, gray dots are negative, red lines are fluorescence threshold values, and A01, B01, C01, D01, E01, F01 and A04 are sequentially indicated from left to right as 56 ℃, 56.9 ℃, 58.1 ℃, 59 ℃, 59.6 ℃ and 60 ℃ and the separation state of the negative droplet and the positive droplet of water. As can be seen from FIG. 1A, the nib8 gene shows good amplification patterns at 6 temperatures, and as the annealing temperature increases, the negative microdroplets and the positive microdroplets of the nib8 gene are more clearly distinguished, and the amplification becomes more and more specific, however, the amplification efficiency of the internal reference gene in FIG. 1B is not greatly changed as the annealing temperature increases. Therefore, 59 ℃ was finally used as the optimal annealing temperature for the nib8 gene and the reference gene to exhibit good amplification effects.
Example 3
Sensitivity (detection lower limit) and accuracy test results
According to the method of example 1, the concentrations of the DNA template in step 1.3, 20ng (A01), 40ng (B01), 160ng (C01) and 320ng (D01), and water are used as a comparative example, and the reaction thermogram of the digital PCR test result is shown in FIG. 2, FIG. 2 shows the digital PCR test result under different template concentrations of nib8 gene and internal reference gene, A, B is the reaction thermogram, A is the nib8 gene detection result, and B is the internal reference gene. The blue dots are positive, the gray dots are negative, the red line is the fluorescence threshold limit, and the template amount gradient and water are 20ng (A01), 40ng (B01), 160ng (C01) and 320ng (D01) from left to right.
In FIG. 2, it can be seen from Panel A that the nib8 gene amplified a good droplet separation at 4 different template concentrations, and the number of droplets increased with increasing concentration. However, in panel B, the reference gene increased with the sample concentration, and at 160ng template concentration, the positive and negative droplets were not separated significantly, and at 320ng template concentration, the phenomenon became more significant. The higher the template concentration, the poorer the amplification effect of the system, and the droplets could not be separated significantly, while the number of droplets was small when the template concentration was 20ng, and the amplification effect could not be seen significantly, so that 40ng was finally selected as the optimal template concentration.
Example 4
6 transgenic wheat lines transformed with nib8 (designated lines 12, 16, 17, 23, 29 and 30, respectively) were selected for copy number analysis according to the detection method described in example 1. (the reaction system and the detection conditions corresponding to the reaction program are shown in Table 2 and Table 3 in 1.3.3 of example 1) the results are shown in detail in FIGS. 3 and 4, Table 4 shows the results of the lower limit assay of the digital PCR assay, FIG. 3 shows the results of the test droplets of 6 nib8 strains, from left to right, strains 12, 16, 17, 23, 29 and 30, respectively, negative control and blank control. From FIG. 3, it can be seen that the number of positive droplets is clearly distinguished from the number of negative droplets, indicating that the system is stable. As can be seen from Table 4, there are three lines in the digital PCR detection results, which are one copy and can be used for further gene function analysis.
TABLE 4 digital PCR detection Low Limit assay results
Example 5
Comparison of different detection methods
6 transgenic wheat lines (named lines 12, 16, 17, 23, 29 and 30) transformed with nib8 gene were selected and subjected to copy number analysis according to the detection method, dye method qPCR and probe method qPCR, respectively, in the example. The results are shown in Table 7.
The reaction system for detecting copy number by dye method qPCR is shown in Table 5:
TABLE 5 dye method qPCR copy number detection reaction system
The reaction system for detecting copy number by probe qPCR is shown in Table 6:
reaction system for detecting copy number by probe method qPCR (quantitative polymerase chain reaction) of Table 6
It can be seen that the digital PCR result is consistent with the real-time PCR analysis result by the dye method and the probe method. FIG. 4 shows that the results of the transgenic nib8 wheat 16 line southern blot are also consistent with the results of the digital PCR, and are both copies.
TABLE 7 comparison of the results of the probe method and the dye method with the digital PCR
With the combination of the above embodiments, compared with the prior art, the qRT-PCR analysis method has more complicated steps, firstly, a standard DNA sample needs to be prepared, then, a standard curve is constructed by using the sample, the establishment of the standard curve also relates to the exploration and optimization of a system, a large amount of time and energy are consumed, and the required test period is longer; in addition, quantification by means of a standard curve is itself a relative quantitative method, so the test results are not accurate. The traditional Southern blot method not only has large demand on DNA and complex test operation steps, but also has higher technical requirements on personnel operation. The digital PCR (dPCR) of the invention can realize sensitive and accurate absolute quantification of the DNA sample without a standard curve. A PCR reaction is divided into a number of independent reactions, each of which has a positive or negative signal. The number of DNA molecules in the original sample was directly calculated from the number of positive and negative reactions using Poisson's statistics. In the research, the requirement of a digital PCR template is low, and compared with the sensitivity of real-time PCR of 0.1 pg. mu.L, the sensitivity of the digital PCR reaches 0.001 pg. mu.L, which is more than 100 times of the sensitivity of the real-time PCR. Compared with Southern, the digital PCR method has short test period, simple operation and high detection flux.
The detection system established by the invention can detect hundreds of samples in a short time, thereby greatly improving the working efficiency. The copy number method established in the present invention can detect not only foreign genes. The copy number of the endogenous gene can also be detected. Therefore, the method has the advantages of rapidness, flexibility, high efficiency, small sample amount and the like, and has wide application prospect in various fields of gene expression research, genome copy number identification, cancer marker rare mutation detection, transgenic component identification and the like.
The present invention has been described in detail above. It will be apparent to those skilled in the art that the invention can be practiced in a wide range of equivalent parameters, concentrations, and conditions without departing from the spirit and scope of the invention and without undue experimentation. While the invention has been described with reference to specific embodiments, it will be appreciated that the invention can be further modified. In general, this application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice within the art to which the invention pertains. The use of some of the essential features is possible within the scope of the claims attached below.
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Claims (8)
1. A digital PCR detection method for detecting the copy number of a wheat gene is characterized by comprising the following steps:
1) extracting the genomic DNA of the wheat to be detected;
2) carrying out enzyme digestion treatment on the genomic DNA extracted in the step 1) by using restriction enzyme;
3) carrying out microdroplet digital PCR reaction;
4) and after the PCR reaction is finished, determining the number of positive droplets and negative droplets of each hole of the sample of the PCR reaction plate, and obtaining the gene copy number of the wheat to be detected.
2. The method for detecting the copy number of nib8 gene in wheat according to claim 1, wherein the step 1) is performed by combining a CTAB method and a full-automatic nucleic acid extractor extraction method to detect the genomic DNA of wheat.
3. The method for detecting the copy number of nib8 gene in wheat according to claim 1, wherein in step 2), the restriction enzyme is EcoRI.
4. The method for detecting the copy number of nib8 gene in wheat according to claim 2, wherein the digestion treatment in step 2) is carried out overnight at 37 ℃, the enzyme is inactivated by treatment for 30min at 65 ℃, and the amount of restriction endonuclease is 1 unit of EcoRI added per microgram of wheat genomic DNA.
5. The method for detecting the copy number of nib8 gene in wheat according to claim 2, wherein the PCR reaction system in step 3) is: 2 XddPCRSuperMix 10. mu.l, 500nmol/L of target gene forward primer 1. mu.L, 500nmol/L of target gene reverse primer 1. mu.L, 500nmol/L of reference gene forward primer 1. mu.L, 500nmol/L of reference gene reverse primer 1. mu.L, 250nmol/L of target gene probe 0.5. mu.l, 250nmol/L of reference gene probe 0.5. mu.l, wheat genome DNA template 5. mu.l, total volume 20. mu.l.
6. The method of claim 5, wherein: the concentration of the wheat genome DNA template is 20 ng/. mu.L.
7. The method of claim 4, wherein: the forward primer of the target gene is Nib8-5-F, and the Nib8-5-F is a single-stranded DNA molecule shown in a sequence 1; the reverse primer of the target gene is Nib8-5-R, and the Nib8-5-R is a single-stranded DNA molecule shown in a sequence 2; the target gene probe is Nib8-5-P, and the Nib8-5-P is a single-stranded DNA molecule shown in a sequence 3; the reference gene forward primer is PINb-D1b-F, and the PINb-D1b-F is a single-stranded DNA molecule shown as a sequence 4; the reference gene reverse primer is PINb-D1b-R, and the PINb-D1b-R is a single-stranded DNA molecule shown in a sequence 5; the reference gene probe is PINb-D1b-P, and the PINb-D1b-P is a single-stranded DNA molecule shown in a sequence 6.
8. The method according to any one of claims 1 to 7, wherein the PCR reaction conditions in step 3) are: at 95 ℃ for 10 min; at 94 ℃, 3s, 59 ℃, 1min, for 40 cycles; at 98 deg.C for 10 min.
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