CN106636340B - Detection method and reagent for transgenic corn strain VCO-01981-5 - Google Patents
Detection method and reagent for transgenic corn strain VCO-01981-5 Download PDFInfo
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Abstract
The application discloses a method and a reagent for detecting a transgenic corn strain VCO-01981-5. The detection method comprises the steps of adding mineral oil into a PCR reaction system, uniformly mixing, transferring to a microdroplet generator to generate microdroplets, then carrying out PCR reaction, reading amplification signals of each microdroplet after the PCR reaction is finished, and calculating the number of target DNA molecules of a sample to be detected by using QuantaSoft software; the PCR reaction system comprises strain VCO-01981-5 and HMG gene specific primers and probes, wherein the two probes are respectively marked by FAM and HEX fluorescence. According to the quantitative detection method, endogenous gene HMG and strain specific primer probes which are matched for use are designed, the number of molecules of the endogenous gene HMG and the strain specific primer probes in a corn genome is respectively quantified by adopting a double-channel method, the absolute content of the VCO-01981-5 in the transgenic corn strain is calculated, and the quantitative detection method has important significance in research on transgenic components and port transgenic detection in China.
Description
Technical Field
The application relates to the field of transgenic corn detection, in particular to a quantitative detection method and a quantitative detection reagent for a transgenic corn strain VCO-01981-5.
Background
Since the commercial planting of transgenic crops in 1996, the planting area of transgenic crops in the world has been rapidly developed. By 2013, the planting area of the global transgenic crops reaches about 1.75 hundred million hectares, which is increased by 3% compared with 2012. According to the planting area statistics, about 81% of soybeans, 35% of corns, 30% of rapes and 81% of cotton are transgenic products all around the world. The countries with transgenic crop planting areas ranked in the top 6 were the united states, brazil, argentina, canada, india and china. Along with the rapid development of transgenic crops, the food safety problem of the transgenic crops has been a hot point discussed for many years and directly influences the international trade and the public acceptance degree of the transgenic products. The current focus of attention has been shifted from 'whether the transgenic crop is the transgenic crop' to 'which transgenic crop components and respective content are contained', in order to meet the requirements of public right to know, management level and detection technical capability, a plurality of countries have been issued with transgenic limit regulations, for example, the European Union regulates that the content of the transgenic crop exceeds 0.9%, and identification is required; whereas the current standard in japan is 5%. The current standard of China is the most strict, zero tolerance is applied to a transgenic product, and identification is needed only when a transgenic component is detected in the product. In consideration of objective reality such as low-level mixing of transgenes and the like, the current 'zero tolerance' policy is too harsh from the technical law enforcement perspective, so that reasonable transgene threshold values are put forward for national policy departments from the practical aspects of the transgene detection level of each port in China; the method is an accurate quantitative detection method for developing transgenic components.
Transgenic corn VCO-01981-5 is a new line of glyphosate-tolerant herbicides developed by the company genetic s.a. (joint venture corporation of Group LIMAGRAIN and Group KWS), headquartered in paris, and approved for commercial production and utilization in the united states and canada in 2013 and 2014, respectively. The strain is not approved by the Ministry of agriculture of China, and no corresponding strain specificity qualitative or quantitative detection method exists in China and China. Therefore, the research on an effective transgenic corn VCO-01981-5 detection method is urgently needed.
Disclosure of Invention
The application aims to provide a method and a reagent for detecting a transgenic corn strain VCO-01981-5.
In order to achieve the purpose, the following technical scheme is adopted in the application:
the application discloses a detection method of a transgenic corn strain VCO-01981-5, which comprises the steps of adding mineral oil into a PCR reaction system, uniformly mixing, transferring the mixture to a droplet generator to automatically generate droplets, then transferring all the droplets to a reaction tube to perform PCR reaction, reading an amplification signal of each droplet after the PCR reaction is finished, and calculating the number of target DNA molecules of a sample to be detected by adopting QuantaSOFT V1.3.2 software; the PCR reaction system is a double real-time fluorescent PCR amplification system and comprises PCR reaction liquid, a strain VCO-01981-5 specific primer group, a strain VCO-01981-5 specific probe, an HMG gene specific primer group, an HMG gene specific probe and DNA of a sample to be detected; the forward primer of the strain VCO-01981-5 specific primer group is a sequence shown in Seq ID No.1, the reverse primer of the strain VCO-01981-5 specific primer group is a sequence shown in Seq ID No.2, the strain VCO-01981-5 specific probe is a sequence shown in Seq ID No.3, the forward primer of the HMG gene specific primer group is a sequence shown in Seq ID No.4, the reverse primer of the HMG gene specific primer group is a sequence shown in Seq ID No.5, and the HMG gene specific probe is a sequence shown in Seq ID No. 6;
Seq ID No.1：5’-aaggccttcagtctactcctcggg-3’
Seq ID No.2：5’-ctagcggccgctactcgagggattt-3’
Seq ID No.3：5’-cgtcaccaagaagatcagtactcaaacac-3’
Seq ID No.4：5’-TTGGACTAGAAATCTCGTGCTGA-3’
Seq ID No.5：5’-GCTACATAGGGAGCCTTGTCCT-3’
Seq ID No.6：5’-CAATCCACACAAACGCACGCGTA-3’
wherein, in the strain VCO-01981-5 specific probe with the sequence shown in Seq ID No.3, the 5 'end is provided with FAM fluorescent group, and the 3' end is provided with BHQ-1 quenching group; in the HMG gene-specific probe having the sequence shown in Seq ID No.6, HEX fluorescent group is provided at the 5 'end, and BHQ-1 quenching group is provided at the 3' end.
It should be noted that the quantitative detection method of the transgenic corn strain VCO-01981-5 of the present application is actually droplet digital PCR. The principle is that the oil-water reaction system is prepared into nearly 20000 water-in-oil small droplets by a droplet generator in a PCR reaction tube, and when the number of molecules of the DNA template to be detected is lower than a certain number, the droplets only contain one molecule of the DNA template and enough other components such as dNTP, Taq DNA polymerase, a probe, a primer and the like required for the fluorescent PCR reaction. After all the microdroplets finish the real-time fluorescent PCR reaction, checking each microdroplet one by one, considering that the real-time fluorescent PCR reaction occurs as long as the microdroplet is detected by a fluorescent signal, detecting the template to be detected which is marked with one molecule, and counting the number of all positive microdroplets to obtain the number of molecules of the DNA template to be detected in the sample. The method adopts a double-channel method, namely, different fluorescent labels are adopted for a corn strain VCO-01981-5 specific probe and a corn endogenous gene specific probe respectively, and the number of molecules of endogenous genes and strain specific sequences is simultaneously measured in the same PCR reaction system. Wherein, the endogenous gene HMG detected by the application has only one copy in the corn genome, and the specific sequence of the corn strain VCO-01981-5 also has only one copy; therefore, the absolute content of the transgenic corn strain VCO-01981-5 can be calculated by calculating the ratio of the number of strain-specific sequences and HMG sequence molecules in the sample.
Therefore, the detection method further comprises the step of calculating the absolute content of the VCO-01981-5 of the transgenic corn strain according to the ratio of the VCO-01981-5 molecular number of the transgenic corn strain to the HMG gene molecular number after respectively calculating the molecular number of the VCO-01981-5 and the HMG gene molecular number of the transgenic corn strain by using QuantaSOFT V1.3.2 software.
The application also discloses a reagent for quantitative detection of the transgenic corn strain VCO-01981-5, which comprises a first primer probe combination and a second primer probe combination, wherein the first primer probe combination is used for specific detection of the strain VCO-01981-5, and the second primer probe combination is used for specific detection of HMG genes; in the first primer probe combination, a forward primer is a sequence shown in Seq ID No.1, a reverse primer is a sequence shown in Seq ID No.2, a probe is a sequence shown in Seq ID No.3, the 5 'end of the probe is provided with a FAM fluorescent group, and the 3' end of the probe is provided with a BHQ-1 quenching group; in the second primer probe combination, the forward primer is a sequence shown in Seq ID No.4, the reverse primer is a sequence shown in Seq ID No.5, the probe is a sequence shown in Seq ID No.6, the 5 'end of the probe is provided with a HEX fluorescent group, and the 3' end of the probe is provided with a BHQ-1 quenching group.
It should be noted that the reagent of the present application is actually the strain VCO-01981-5 specific detection primer probe and HMG gene specific detection primer and probe used in the quantitative detection method of transgenic maize strain VCO-01981-5. It is understood that the primers and probes of the present application are designed for digital PCR in micro-drop form, and can be prepared separately to be used as the corresponding detection reagent of strain VCO-01981-5.
The application also discloses application of the reagent in preparation of a transgenic corn detection kit or detection equipment.
The application also discloses a transgenic corn detection kit, which contains the reagent.
Preferably, the kit of the present application quantitatively detects the transgenic corn strain VCO-01981-5 by using the quantitative detection method of the present application.
Due to the adoption of the technical scheme, the beneficial effects of the application are as follows:
according to the detection method of the transgenic corn strain VCO-01981-5, an endogenous gene HMG (human growth factor) specific primer probe and a strain VCO-01981-5 specific primer probe which are used in a matched mode are designed, the endogenous gene HMG specific primer probe and the strain VCO-01981-5 specific primer probe are used in a matched mode, the number of molecules of the endogenous gene HMG specific primer probe and the strain VCO-01981-5 in a corn genome is respectively quantified by adopting a double-channel method, and the absolute content of the transgenic corn strain VCO-01981-5 is. The quantitative detection method is different from the conventional relative quantification of real-time fluorescence PCR, can accurately quantify the transgenic corn strain VCO-01981-5 in the sample to be detected, and has important significance on the research of transgenic components and the detection of port transgenes in China.
Drawings
FIG. 1 is a graph showing the output results of a specific detection FAM channel in the example of the present application;
FIG. 2 is a graph showing the output results of the specific detection of HEX channels in the examples of the present application;
FIG. 3 is a graph showing the output results of the stability test FAM channel in the example of the present application;
FIG. 4 is a graph of the output of the HEX channel of the stability test in the example of the present application;
FIG. 5 is a graph of fitted standard curves for HEX channel data for gradient dilution testing in an embodiment of the present application;
fig. 6 is a plot of fitted standards for FAM channel data for gradient dilution detection in an embodiment of the present application.
Detailed Description
The transgenic corn line VCO-01981-5 is a newly developed transgenic corn line in recent years, and no corresponding qualitative or quantitative detection method exists at present. Therefore, the detection method of the transgenic corn strain VCO-01981-5 is researched and provided firstly, not only can the strain VCO-01981-5 be detected qualitatively, but also accurate absolute quantification can be carried out on the strain VCO-01981-5.
The detection method is different from the conventional methods such as general PCR, real-time fluorescence PCR, PCR-DHPLC and the like used in the detection of the transgenic corn strain; generally, PCR can only be detected qualitatively, and although real-time fluorescence PCR can be detected by a standard substance diluted in a gradient manner, a standard curve is established, and relative quantification is carried out through the standard curve; however, absolute quantitative detection is not possible. With the increasing popularity of transgenic crops, China is in accordance with the policy of 'zero tolerance' of transgenes, and the existing relatively quantitative detection method cannot meet the use requirements of port transgenic detection in China. Therefore, the application utilizes the advanced digital PCR detection technology to firstly research and provide a quantitative detection method of the transgenic corn strain VCO-01981-5. In the quantitative detection method, two sets of primer probes matched with the endogenous gene and the strain gene are simultaneously designed, the molecular number of specific sequences of the endogenous gene HMG and the strain VCO-01981-5 is respectively statistically analyzed by using two fluorescence channels through double digital PCR amplification, and the absolute content of the transgenic corn strain VCO-01981-5 is calculated according to the ratio of the molecular number of the transgenic corn strain VCO-01981-5 to the molecular number of the HMG gene.
The present application is described in further detail below with reference to specific embodiments and the attached drawings. The following examples and figures are merely illustrative of the present application and are not to be construed as limiting the present application.
Examples
Materials and methods
1. Test material
This example was tested using DNA samples of transgenic maize lines VCO-01981-5, MIR162, MON89034, Bt176, Bt11, MON88017, MON810, MON863, T25, MIR604 and ES3272, respectively. The DNA samples are provided and preserved by Shenzhen entry and exit inspection and quarantine bureau animal and plant inspection and quarantine technical center.
2. Primer and probe design
In the embodiment, specific detection primers and probes are designed according to the corn endogenous gene HMG and the specific sequence of the transgenic corn strain VCO-01981-5, and the two probes are respectively marked by different fluorophores. The primers and probes were synthesized and modified by Biotechnology engineering (Shanghai) Inc. The primer and probe sequences designed in this example are detailed in Table 1.
TABLE 1 primers and probes specific for the endogenous Gene and line VCO-01981-5
| Name (R) | Sequence (5 '-3') | Seq ID No. |
| VCO-01981-5-F | aaggccttcagtctactcctcggg | 1 |
| VCO-01981-5-R | ctagcggccgctactcgagggattt | 2 |
| VCO-01981-5-P | cgtcaccaagaagatcagtactcaaacac | 3 |
| HMG-F | TTGGACTAGAAATCTCGTGCTGA | 4 |
| HMG-R | GCTACATAGGGAGCCTTGTCCT | 5 |
| HMG-P | CAATCCACACAAACGCACGCGTA | 6 |
Wherein, FAM fluorescent group is carried out on the 5 'terminal of the VCO-01981-5-P probe, and BHQ-1 quenching group is carried out on the 3' terminal; HEX fluorescent group is carried out on the 5 'terminal of the HMG-P probe, and BHQ-1 quenching group is carried out on the 3' terminal.
3. Dual digital PCR reaction systems and conditions
The PCR reaction system of this example amounted to 20. mu.L, including 2 × ddPCR Super Mix 10. mu.L, 3.6. mu. mol/L of corn line-specific probe VCO-01981-5-P1. mu.L, 6. mu. mol/L of corn line forward primer VCO-01981-5-F1. mu.L, 6. mu. mol/L of corn line reverse primer VCO-01981-5-R1. mu.L, 3.6. mu. mol/L of endogenous gene probe HMG-P1. mu.L, 6. mu. mol/L of endogenous gene forward primer HMG-F1. mu.L, 6. mu. mol/L of endogenous gene reverse primer HMG-R1. mu.L, 2. mu.L of DNA template of about 15 ng/. mu.L, and supplemented with ddH2O to 20. mu.L.
After 20 mul of reaction liquid is prepared, the reaction liquid is evenly mixed with 70 mul of mineral oil and transferred to a microdroplet generator to automatically generate microdroplets; carefully transferring all the generated droplets into a 96-well reaction plate PCR reaction tube; and then the 96-hole reaction plate is sealed on a membrane sealing instrument and is placed on a common PCR instrument for PCR reaction.
The reaction conditions are as follows: pre-denaturation at 95 ℃ for 10 min; then 40 cycles are entered: denaturation at 95 ℃ for 15s, and annealing at 55.7 ℃ for 1 min; the enzyme was heat-inactivated at 98 ℃ for 10min after the end of the cycle. And finishing the amplification.
After amplification was complete, the 96-well plate was placed in a microdroplet reader to read the signal and the experimental data was analyzed using QuantaSoft V1.3.2 software. Specifically, the microdroplet fluorescence detector application software is opened, the 96-hole reaction plate after the PCR reaction is finished is directly inserted into the fluorescence detector, the device automatically detects the fluorescence condition of all microdroplets in each PCR reaction tube, namely the PCR reaction condition, and finally the software directly gives the number of the DNA sequence molecules to be detected according to the Poisson's distribution law. In this example, the number of molecules of transgenic maize line VCO-01981-5 was obtained according to the FAM fluorescence channel, and the number of molecules of endogenous gene HMG was obtained according to the HEX fluorescence channel.
The absolute content of the transgenic corn strain VCO-01981-5 can be calculated according to the ratio of the number of the strain VCO-01981-5 molecules to the number of the endogenous gene HMG molecules.
4. Quality control
A control is required to be set in each batch (time) of quantitative detection experiments, and the setting of the control is in accordance with the regulation in GB/T19495.2-2004.
The number of microdroplets in each PCR reaction tube is not less than 12000; the number of molecules added to the maize genome is not more than 5 times the number of microdroplets, about 2.5pg per maize genomic DNA molecule, and therefore, the amount added to the maize genome is not more than 12000 × 5 × 2.5 ═ 150000pg, i.e., 150 ng.
Theoretical detection results of a non-transgenic sample nucleic acid extraction control, a PCR blank control and a negative control should be zero. However, considering the ultra-sensitivity of digital PCR, especially the zero detection result of the non-transgenic sample nucleic acid extraction control is sometimes difficult to achieve, thus allowing the occurrence of a very small number of positive droplets, but the number of positive droplets should be less than 1/4000, and based on the experience, 12000 droplets cannot exceed three positive droplets, and the effective positive detection result is at least three times that of the control positive droplets, i.e., at least 9 positive droplets.
The relative difference of the contents of the transgenic components of the test results of the DNA samples extracted twice is less than or equal to 25 percent.
Wherein, a is the detection result of the first extraction of the DNA sample; b is the detection result of the second extraction of the DNA sample; m is the average of a and b.
For DNA samples extracted from the same batch, the deviation of the results of the three-time repeated PCR test should be less than or equal to 15%.
If one of the above quality control conditions is not satisfied, the test result should be discarded and the test should be redone from the preparation of the test sample.
5. Specificity detection
This example was tested specifically with a positive sample of the DNA sample of transgenic maize line VCO-01981-5 and negative samples of transgenic maize lines MIR162, MON89034, Bt176, Bt11, MON88017, MON810, MON863, T25, MIR604 and ES3272, with a water blank set.
6. Stability test
This example used a DNA sample of transgenic corn line VCO-01981-5 diluted to a working concentration of 20 ng/. mu.L, tested in 3 replicates using the dual digital PCR reaction system and conditions of this example, and a DNA sample of transgenic corn line MIR162 was set as a negative control and a water blank was set. The loading amount of the DNA sample in this example was 50ng, and the rest was the same as "3. double digital PCR reaction system and conditions".
7. Test for detecting Linear Range
The linear range of the present example refers to the range in which the digital PCR detection system can perform absolute quantitative detection of the transgenic components. Since this example is an absolute quantitative assay based on digital PCR, determining the linear range of the assay method is of great significance in defining the range of applicability of the assay method.
In this example, DNA samples of transgenic maize line VCO-01981-5 were diluted in 7 gradients to 25 ng/. mu.L, 5 ng/. mu.L, 1 ng/. mu.L, 0.2 ng/. mu.L, 0.04 ng/. mu.L, 0.008 ng/. mu.L and 0.0016 ng/. mu.L, tested at 2. mu.L loading according to the "3. Dual digital PCR reaction System and conditions" of this example, with 3 replicates per concentration, and a DNA sample of line MIR162 as a negative control, and a water blank.
8. Quantitative detection limit and detection limit verification of corn VCO-01981-5 digital PCR
In the Detection method Of this example, the Limit Of quantitative Detection (LOQ) and the Limit Of Detection (LOD) are respectively defined as the lowest copy number at which stable quantitative Detection or Detection can be performed.
The LOQ verification was performed by diluting the DNA sample with the lower limit gradient of detection of "7. detection Linear Range assay". Specifically, the DNA sample was subjected to ten-fold digital PCR verification, the copy number of the endogenous gene HMG and strain VCO-01981-5 specific sequence per unit volume of each duplicate experiment was calculated, and the average value and RSD value were calculated.
Similarly, the LOD was verified by diluting the DNA sample with a gradient of the lower limit of detection of "7. detection Linear Range assay". Specifically, the DNA sample was subjected to digital PCR verification of 10 replicates, and the copy number of the endogenous gene HMG and strain VCO-01981-5-specific sequence per unit volume of each replicate and the average thereof were calculated.
9. Precision experiment of corn VCO-01981-5 digital PCR
Precision refers to the standard deviation of the test results obtained by the same method, the same test item, in the same laboratory, and by the same person, within a short time interval, under reproducible conditions, using the same instrumentation.
In this example, precision verification was carried out using DNA samples at 10-fold concentrations of the LOQ cutoff value for the foreign gene and the LOQ cutoff value, 6 replicates were made for each sample, the copy number of the specific sequences of the endogenous gene HMG and strain VCO-01981-5 per unit volume of each replicate experiment was calculated, and the average value and RSD value were calculated.
10. Accuracy experiment of corn VCO-01981-5 digital PCR
Accuracy is defined as the degree of correspondence between the test result and the set of public values. In the experiment of this example, 3 groups of positive DNA samples with LOQ value, LOQ5 times and LOQ 25 times were sampled for accuracy verification. Three replicate digital PCR experiments were performed on the above 3 sets of DNA samples. Calculating the ratio of the copy number of the specific sequence of the strain VCO-01981-5 to the copy number of the endogenous gene HMG, comparing the determined ratio with a theoretical ratio, calculating the deviation of the ratio, and analyzing the accuracy of the detection method.
Second, results and analysis
1. Specific detection results
The specific detection results are shown in fig. 1 and fig. 2, wherein fig. 1 shows the fluorescence signal detected by the FAM channel, i.e. the amplification signal of the strain specific probe VCO-01981-5-P; FIG. 2 shows the fluorescence signal detected by the HEX channel, i.e.the amplification signal of the endogenous gene-specific probe HMG-P; in fig. 1 and 2, reaction wells a01-B02 are the detection results of transgenic corn lines MIR162, MON89034, Bt176, Bt11, MON88017, MON810, MON863, T25, MIR604 and ES3272, respectively, and reaction well E02 is the detection result of VCO-01981-5.
The results show that in the detection method of the example, only the strain VCO-01981-5 of the 11 transgenic maize strains of the FAM channel has amplification signals, and negative samples of other 10 transgenic maize strains have no amplification signals. In the HEX channel, 11 transgenic maize lines all have amplification signals. The above results indicate that the line VCO-01981-5 primer and probe specificity results are good.
2. Stability test
A DNA sample of line VCO-01981-5 at a concentration of 20 ng/. mu.L was subjected to stability test thermograms for 3 replicates as shown in FIGS. 3 and 4, and data analysis of the experimental results is shown in Table 2. Fig. 3 is a result graph of the FAM channel, fig. 4 is a result graph of the channel HEX, and according to the heat spot diagram obtained by the reaction, as shown in fig. 3, the difference of fluorescence signals between the negative point and the positive point obtained by the digital PCR is significant, the tailing phenomenon is not serious, and the negative reaction point and the positive reaction point can be distinguished by setting a uniform threshold. The final results of this step are shown in Table 2, and the actual copy numbers of the endogenous gene probes in the three repeated reactions are 13160, 13500 and 137200, respectively, the average copy number is 13460, and the Relative Standard Deviation (RSD) is 2.09%, which is within an acceptable range; the actual copy numbers of the three repeated reactions of the foreign gene are 4660, 4480 and 4740 respectively, the average copy number is 4626, and the RSD is 2.87%, which are within an acceptable range. Therefore, the primer probes for the endogenous gene and the exogenous gene of this example were excellent in amplification stability, and the positive sample was stably usable. It should be noted that RSD equal to or less than 10% falls within the acceptable range, and in this example, the RSD is much less than this value.
Table 2 strain VCO-01981-5 stability detection results
In table 2, the detection channel FAM is the detection result of the strain VCO-01981-5 specific probe, and the detection channel HEX is the detection result of the endogenous gene HMG specific probe.
3. Test for detecting Linear Range
The 7-step dilutions of this example were 25 ng/. mu.L, 5 ng/. mu.L, 1 ng/. mu.L, 0.2 ng/. mu.L, 0.04 ng/. mu.L, 0.008 ng/. mu.L and 0.0016 ng/. mu.L, and in the case of a sample size of 2. mu.L, they corresponded to 19002, 3800, 760, 152, 30, 6 and 1.2 copies of maize genomic DNA, respectively. The DNA templates of the above gradient dilution groups were subjected to digital PCR detection, 3 replicates were set for each concentration gradient group, the experimental detection results are shown in Table 3, and the data fitting standard curve in the linear range is shown in FIGS. 5 and 6. FIG. 5 is a graph showing the relationship between the amount of DNA sample used and the copy number of the specific sequence of the tested strain, and it is found that the copy number of the specific sequence of the strain measured by the method of this example is highly positively correlated with the amount of the DNA sample in the range of 50ng-0.08ng of the DNA sample, the correlation coefficient is more than 0.99, and the fitting curve equation is y-4.673 x-0.6313, which indicates that the quantitative linear range of the specific sequence of the strain in the method of this example is between 4.8-4666.6 copies and spans three orders of magnitude. FIG. 6 is a graph showing the relationship between the amount of DNA sample and the copy number of the endogenous gene to be detected, and it is found that the copy number of the endogenous gene shows a good linear relationship with the amount of the DNA sample in the interval of 3.8-13646.6, the correlation coefficient is above 0.99, and the fitting curve equation is that y is 13.669x-2.3115, which indicates that the quantitative linear range of the sequence of the endogenous gene in the method of the present embodiment is between 3.8-13646.6 copies and spans four orders of magnitude. The research results show that the digital PCR method established by the embodiment has wide quantitative range, high quantitative precision and good result repeatability.
Table 3 Linear range verification results of strain VCO-01981-5 digital PCR
4. Quantitative detection limit and detection limit verification result of corn VCO-01981-5 digital PCR
In this example, LOQ was verified using a DNA sample load of 0.08ng, and LOD was verified using a DNA sample load of 0.016 ng.
LOQ verification results of digital PCR of strain VCO-01981-5 in Table 4
LOD verification results of digital PCR of strain VCO-01981-5 in Table 5
The results of ten repeated digital PCR tests on the verified DNA sample of LOQ are shown in Table 4, and the results show that the RSD of 10 parallel tests of the DNA sample is 24%, which meets the requirement of less than 25%, thus the test method of the embodiment can stably and quantitatively detect the sample with the copy number of the exogenous gene of 5 per unit volume.
The results of ten repeated digital PCR tests on the DNA samples for LOD verification are shown in Table 5, and the results show that the positive detection rate of the endogenous genes in ten parallel samples is 10, which meets the requirement that the LOD quality control is not less than 9, and the method can be used for stably detecting the samples with the endogenous gene copy number of 4 in unit volume.
5. Precision experimental result of corn VCO-01981-5 digital PCR
In this example 6 replicates were run using DNA sample loads of 0.08ng and 0.8ng, respectively. As shown in Table 6, it can be seen that, by comprehensively analyzing the results of the DNA samples with the LOQ critical point and the critical point 10 times, the RSD values in the sets obtained by the DNA samples with the two concentrations are 22.78% and 10.29%, respectively, which meets the requirement of the whole detection method for precision, i.e., the RSD is required to be less than 25%.
Precision results of digital PCR of strain VCO-01981-5 in Table 6
6. Accuracy experimental result of corn VCO-01981-5 digital PCR
In this example, three replicates were run using DNA sample loads of 0.016ng, 0.08ng and 0.4ng, respectively, and the results are shown in Table 7.
TABLE 7 results of the numerical PCR accuracy experiment of corn VCO-01981-5
The results show that the average values of the percent of the measured copies of the DNA standard samples with the sample loading amounts of 0.016ng, 0.08ng and 0.4ng are respectively 50%, 56.6% and 50%, the theoretical percent of the copies of the DNA standard samples is 50%, the calculated detection deviation is respectively 0.00%, 13.20% and 0.00%, and the three groups of deviations meet the requirement of less than 25%. Therefore, the method can accurately perform absolute quantitative detection on the BVLA430101 strain.
The foregoing is a more detailed description of the present application in connection with specific embodiments thereof, and it is not intended that the present application be limited to the specific embodiments thereof. For those skilled in the art to which the present application pertains, several simple deductions or substitutions may be made without departing from the concept of the present application, and all should be considered as belonging to the protection scope of the present application.
SEQUENCE LISTING
<110> Shenzhen entry and exit inspection and quarantine bureau animal and plant inspection and quarantine technical center
<120> detection method and reagent for transgenic corn strain VCO-01981-5
<130>16I22568
<160>6
<170>PatentIn version 3.3
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<211>24
<212>DNA
<213> Artificial sequence
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aaggccttca gtctactcct cggg 24
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<212>DNA
<213> Artificial sequence
<400>2
ctagcggccg ctactcgagg gattt 25
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<212>DNA
<213> Artificial sequence
<400>3
cgtcaccaag aagatcagta ctcaaacac 29
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<213> Artificial sequence
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ttggactaga aatctcgtgc tga 23
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<210>6
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<213> Artificial sequence
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caatccacac aaacgcacgc gta 23
Claims (5)
1. A detection method of a transgenic corn strain VCO-01981-5 is characterized by comprising the following steps: adding mineral oil into a PCR reaction system, uniformly mixing, transferring the mixture to a microdroplet generator to automatically generate microdroplets, then transferring all the microdroplets to a reaction tube to perform PCR reaction, reading an amplification signal of each microdroplet after the PCR reaction is finished, and calculating the number of target DNA molecules of a sample to be detected by adopting QuantaSOFT V1.3.2 software;
the PCR reaction system is a double real-time fluorescent PCR amplification system and comprises PCR reaction liquid, a strain VCO-01981-5 specific primer group, a strain VCO-01981-5 specific probe, an HMG gene specific primer group, an HMG gene specific probe and DNA of a sample to be detected;
the forward primer of the strain VCO-01981-5 specific primer set is a sequence shown by Seq ID No.1, the reverse primer of the strain VCO-01981-5 specific primer set is a sequence shown by Seq ID No.2, the strain VCO-01981-5 specific probe is a sequence shown by Seq ID No.3, the forward primer of the HMG gene specific primer set is a sequence shown by Seq ID No.4, the reverse primer of the HMG gene specific primer set is a sequence shown by Seq ID No.5, and the HMG gene specific probe is a sequence shown by Seq ID No. 6;
Seq ID No.1：5’-aaggccttcagtctactcctcggg-3’
Seq ID No.2：5’-ctagcggccgctactcgagggattt-3’
Seq ID No.3：5’-cgtcaccaagaagatcagtactcaaacac-3’
Seq ID No.4：5’-TTGGACTAGAAATCTCGTGCTGA-3’
Seq ID No.5：5’-GCTACATAGGGAGCCTTGTCCT-3’
Seq ID No.6：5’-CAATCCACACAAACGCACGCGTA-3’
wherein, in the strain VCO-01981-5 specific probe with the sequence shown in Seq ID No.3, the 5 'end is provided with FAM fluorescent group, and the 3' end is provided with BHQ-1 quenching group; in the HMG gene specific probe with the sequence shown in Seq ID No.6, the 5 'end is provided with a HEX fluorescent group, and the 3' end is provided with a BHQ-1 quenching group;
the detection method further comprises the step of calculating the absolute content of the VCO-01981-5 of the transgenic corn strain according to the ratio of the VCO-01981-5 of the transgenic corn strain to the HMG gene molecule number after the QuantaSOFT V1.3.2 software is adopted to calculate the molecule number of the VCO-01981-5 and the HMG gene molecule number of the transgenic corn strain respectively.
2. A reagent for quantitative detection of transgenic corn strain VCO-01981-5 is characterized in that: the reagent comprises a first primer probe combination and a second primer probe combination, wherein the first primer probe combination is used for the specific detection of the strain VCO-01981-5, and the second primer probe combination is used for the specific detection of HMG genes;
in the first primer probe combination, a forward primer is a sequence shown in Seq ID No.1, a reverse primer is a sequence shown in Seq ID No.2, a probe is a sequence shown in Seq ID No.3, the 5 'end of the probe is provided with a FAM fluorescent group, and the 3' end of the probe is provided with a BHQ-1 quenching group;
in the second primer probe combination, the forward primer is a sequence shown in Seq ID No.4, the reverse primer is a sequence shown in Seq ID No.5, the probe is a sequence shown in Seq ID No.6, the 5 'end of the probe is provided with a HEX fluorescent group, and the 3' end of the probe is provided with a BHQ-1 quenching group.
3. Use of the reagent according to claim 2 in the preparation of a transgenic corn assay kit or assay device.
4. A transgenic corn detection kit is characterized in that: the kit contains the reagent of claim 2.
5. The transgenic corn assay kit of claim 4, wherein: the kit adopts the detection method of claim 1 to quantitatively detect the transgenic corn strain VCO-01981-5.
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| CN107142322A (en) * | 2017-06-22 | 2017-09-08 | 中华人民共和国黄埔出入境检验检疫局 | Transgenic corns MON87403 strain specificities real-time fluorescent PCR testing primer, probe, method and kit |
| CN109825632A (en) * | 2019-04-03 | 2019-05-31 | 深圳出入境检验检疫局食品检验检疫技术中心 | Transgenic corn lines DAS40278-9 detection method and reagent |
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| CN109852724A (en) * | 2019-04-03 | 2019-06-07 | 深圳出入境检验检疫局食品检验检疫技术中心 | Transgene rape strain OXY235 detection method, reagent and kit |
| CN109825634A (en) * | 2019-04-03 | 2019-05-31 | 深圳出入境检验检疫局食品检验检疫技术中心 | Transgene rape strain DP-073496-4 detection method and reagent |
| CN109811083A (en) * | 2019-04-03 | 2019-05-28 | 深圳出入境检验检疫局食品检验检疫技术中心 | Transgene rape strain MON88302 detection method and reagent |
| CN109913571A (en) * | 2019-04-03 | 2019-06-21 | 深圳出入境检验检疫局食品检验检疫技术中心 | Transgenic corn lines MON87427 detection method and reagent |
| CN109825633A (en) * | 2019-04-03 | 2019-05-31 | 深圳出入境检验检疫局食品检验检疫技术中心 | Reagent, kit and the detection method of transgene rape strain Ms1 detection |
| CN109825635A (en) * | 2019-04-03 | 2019-05-31 | 深圳出入境检验检疫局食品检验检疫技术中心 | Method, reagent and the kit that Transgenic corn lines 98140 detect |
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