Preparation method of mixed standard plasmid, mixed standard plasmid and kit
By preparing and diluting mixed standard plasmids, the problems of plasmid uniformity and stability are solved, the accuracy and consistency of multi-gene detection are ensured, the operation process is simplified, the cost is reduced, and the experimental efficiency is improved.
Patent Information
- Application Number
- CN202510231071.0
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-02-28
- Publication Date
- 2025-07-11
AI Technical Summary
During the preparation of existing mixed standard plasmids, the uniformity of plasmids is difficult to control, and there is a risk of degradation or mutation. The interaction between plasmids leads to structural changes or loss of function, which affects the accuracy and consistency of experimental results, especially in multi-gene detection, and is complex in management.
The target gene fragment with sequence numbers such as SEQ ID NO: 1 to 120 was synthesized with pESI-T vector, insert the EcoRI site, transform E. coli competent cells, screen positive clones, extract plasmids, dissolve and dilute with TE buffer to ensure that each plasmid concentration reaches 109copies/μL, determine the optimal concentration range through 10 times dilution, and extract the plasmid using EZ rotary column plasmid DNA drug preparation kit to ensure the stability and uniformity of the plasmid.
The high concentration uniformity and stability of mixed standard plasmids are achieved, the accuracy and consistency of multi-gene detection is ensured, the experimental cost is reduced, the operation process is simplified, and the experimental efficiency and reliability of results are improved.
Smart Images
Figure CN120290610A_ABST
Abstract
Description
Technical Field
[0001] The present invention relates to the field of preparation of mixed standard plasmids. Background Art
[0002] Standard plasmids are used to generate standard curves and are key tools for achieving accurate quantification of target genes, playing a crucial role in qPCR. By providing stable and reproducible standards, standard plasmids can effectively evaluate the detection sensitivity of qPCR, correct errors between experiments, and evaluate the efficiency of PCR reactions. In addition, standard plasmids can also be used as quality control tools for experiments to help identify and eliminate systematic and accidental errors.
[0003] With the development of HT-qPCR, the demand for multi-gene detection in experiments has increased significantly, thereby significantly increasing the demand for mixed standard plasmids. Mixed standard plasmids can not only generate standard curves for multiple target genes simultaneously, reducing time and workload and improving experimental efficiency, but also ensure that all target genes are quantitatively analyzed under the same reaction conditions, reducing variations in different reaction systems and ensuring the consistency of results. At the same time, the use of mixed standard plasmids simplifies the operation process and reduces experimental costs and resource consumption.
[0004] However, there are still significant challenges in the preparation of existing mixed standard plasmids. First, it is difficult to control the uniformity of each plasmid after mixing, and during the preparation and storage processes, the plasmids may degrade or mutate. Especially when multiple plasmids are mixed, the stability differences of different plasmids increase the complexity of management. In addition, when multiple plasmids coexist, plasmid-plasmid interactions may occur, resulting in structural changes or loss of function, and even affecting their replication and expression. In large-scale experiments, ensuring the consistency and reproducibility of mixed standard plasmids is an even greater challenge. Any minor deviation may lead to instability of the standard curve and affect the accuracy of experimental results. In view of this, designing a standardized preparation process and adopting high-precision monitoring technologies to ensure the consistency and quality of plasmids are technical problems that need to be solved urgently. We have successfully addressed these challenges through an innovative plasmid mixing and dilution optimization scheme, providing a reliable and efficient standard plasmid solution for multi-gene detection.
[0005] At the same time, when some existing mixed plasmids are prepared, each plasmid is dissolved separately and then the dissolved solutions are mixed. The concentration of the dissolved plasmids is low, which is not conducive to subsequent detection. Summary of the Invention
[0006] The object of the present invention is to disclose a method for preparing a mixed standard plasmid for the problems existing in the prior art, and the steps are as follows:
[0007] The target gene fragments with sequence numbers as shown in SEQ ID NO: 1 to 120 and the pESI-T vector were synthesized to obtain 120 synthetic plasmids, and the target gene fragments were inserted into the EcoRI site of the pESI-T vector respectively;
[0008] The synthetic plasmids were dissolved in TE buffer to a final concentration of 25 ng / μL and then transformed into Escherichia coli competent cells to introduce the synthetic plasmids into the cells, obtaining transformation products;
[0009] The transformation products were evenly spread on LB plates containing the corresponding antibiotics; positive clones were screened to obtain 120 positive clones;
[0010] The 120 positive clones were subjected to amplified culture, and plasmids were extracted from the cultures;
[0011] The plasmids were verified;
[0012] The plasmid concentrations were measured to prepare diluted standard plasmids.
[0013] In a specific protocol, the method for extracting plasmids from the cultures was to use an EZ Spin Column Plasmid DNA Purification Kit to extract plasmids.
[0014] In a specific protocol, the specific steps for introducing the synthetic plasmids into the cells were as follows: Take competent cells, dilute and aspirate the plasmids, add them to the competent cells, and mix well in a centrifuge tube; then place the centrifuge tube in an environment at 0 °C; partially immerse the centrifuge tube in a water bath for heat shock; after heat shock, let the centrifuge tube cool down; add medium for recovery culture; after recovery, obtain the transformation products.
[0015] In a specific protocol, the method for extracting plasmids was as follows:
[0016] Add the overnight culture to a centrifuge tube and centrifuge; completely drain the liquid; add 5 ml of Solution I to the pellet, gently mix, and let it stand on ice. Add 5 ml of Solution II to the mixture, gently mix by inverting the tube 4 - 6 times, and then let it stand at room temperature; add 7 ml of Solution III and gently mix; incubate at room temperature; centrifuge; place the column into a collection tube; transfer the above supernatant to the column and let it stand at room temperature for 5 min. Centrifuge at 6000 rpm for 3 - 5 min; discard the effluent in the tube. Add the washing solution to the column and centrifuge for washing; repeat the washing process; discard the effluent in the collection tube; centrifuge for 5 min to remove the residual washing solution; transfer the column to a clean 50 ml microcentrifuge tube; add 500 μl of elution buffer to the central part of the column membrane and incubate for 2 min; centrifuge for 2 min.
[0017] In a specific protocol, the mixed standard plasmid was prepared as follows: A special method was adopted, that is, 120 plasmids were dissolved one by one in the form of dry powder. Specifically, the first plasmid dry powder was dissolved in TE buffer to obtain the first dissolution solution, and then the first dissolution solution and the second plasmid dry powder were dissolved (pour the first dissolution solution into the tube containing the second plasmid dry powder) to obtain the second dissolution solution, and so on, to obtain a high-concentration mixed standard plasmid. This method helps to ensure that the concentration of each plasmid reaches a very high level (10 9 copies / μL), so as to maintain the uniformity and stability of the plasmid during subsequent dilution. TE buffer is used to maintain the stability of the plasmid during the dissolution process, and this choice has significant technical advantages. TE buffer can protect DNA from degradation, especially during long-term storage or multiple dilutions, which helps to maintain the integrity and activity of the plasmid.
[0018] In a specific protocol, the method for preparing the diluted standard plasmid is as follows: The prepared mixed standard plasmid was serially diluted 10-fold to obtain 10 8 -10 copies / μL, and then 10 8 , 10 7 , 10 6 copies / μL were serially diluted two-fold to obtain plasmids with diluted concentrations. Through experiments, the appropriate dilution gradient was finally determined, and within this gradient range, the standard curve performed very well, the plasmid DNA concentration was appropriate, the amplification efficiency was guaranteed, and the reliability of the quantitative results was ensured.
[0019] This application also discloses a mixed standard plasmid, which is characterized in that it includes the gene sequences of SEQ ID NO: 1-120.
[0020] This application also discloses a kit, which includes the above-mentioned mixed standard plasmid.
[0021] This application has determined the optimal concentration range suitable for the mixed standard plasmid to ensure reliable and reproducible quantification of various target genes. By establishing this ideal working range, we aim to provide a standardized and easy-to-apply solution for researchers, thereby further promoting the wide application of high-throughput qPCR technology in various research environments.
[0022] Based on this, for 119 target gene fragments and the bacterial 16S rRNA gene, individual plasmids were constructed separately and then mixed to prepare a mixed standard plasmid. During this process, we systematically tested and optimized the mixed standard plasmid at different dilution gradients. Through this method, we successfully constructed a mixed standard plasmid that can accurately and absolutely quantify both the inserted element containing the target gene fragment and the bacterial 16S rRNA gene, and determined its optimal concentration range for use. This preparation process and usage method ensure the stability and accuracy of each target gene in high-throughput detection, providing a reliable multi-gene quantitative analysis tool. BRIEF DESCRIPTION OF THE DRAWINGS
[0023] Figure 1 are the ordinary qPCR and Ct values of the serially diluted mixed standard plasmid;
[0024] Figure 2 are the high-throughput qPCR and Ct values of the serially diluted mixed standard plasmid;
[0025] Figure 3 is the statistical result of the amplification efficiency of the mixed standard plasmid in ordinary qPCR and HT-qPCR;
[0026] Figure 4 is the result comparison of the quantification of the 16S rRNA gene in the same sample using two kinds of qPCR based on the mixed standard plasmid (Ratio1 is the ratio of the result of HT-qPCR to the result of ordinary qPCR, and Ratio2 is the reciprocal of Ratio1);
[0027] Figure 5 is the schematic diagram of the synthetic plasmid; DETAILED DESCRIPTION OF THE INVENTION
[0028] The present invention will be described in detail below with reference to the accompanying drawings.
[0029] Example 1:
[0030] I. Preparation of the plasmid containing the target gene fragment
[0031] The pESI-T vector was selected, and the target gene fragments shown in SEQ ID NO: 1-120 were inserted at its EcoR I site respectively. All gene sequence information was sent to a biological company for synthesis. Finally, the company delivered 4 μg of the lyophilized plasmid containing the target gene insertion fragment. The lyophilized plasmid was dissolved in TE buffer to a final concentration of 25 ng / μL to prepare a synthetic plasmid solution for subsequent transformation and cloning experiments.
[0032] II. Obtaining positive clones carrying the target gene fragment
[0033] The synthetic plasmid solution was transformed into Escherichia coli competent cells. The specific steps are as follows:
[0034] (1) Take 1 tube of competent cells containing 100 μL from a refrigerator at -80 °C and place it in an ice-water mixture for thawing.
[0035] (2) Dilute the concentration of the synthetic plasmid solution to 10 ng / μL, aspirate 3 μL of the plasmid, and add it to the thawed competent cells. Gently flick the centrifuge tube to mix evenly.
[0036] (3) Then ice-bathe the centrifuge tube for 10 min.
[0037] (4) Heat-shock the above centrifuge tube in a 42 °C water bath for 90 seconds and then quickly ice-bathe for 3 min;
[0038] (6) Subsequently, add 600 μL of LB medium and resuscitate and culture at 37 °C on a shaker (200 rpm) for 45 min.
[0039] (7) After the resuscitation is completed, aspirate 100 μL of the transformation product and evenly coat it on an LB plate containing ampicillin antibiotic;
[0040] (8) Invert the plate and place it in a 37 °C incubator for overnight culture.
[0041] Colony PCR screening of positive clones: The reaction system is 20 μL. Use universal primers M13F and M13R. Dip a toothpick into the colony as the PCR template, and at the same time spot the plate for storage. Detect by agarose gel electrophoresis. When the length of the electrophoresis band is consistent with the target product, culture the corresponding colony in LB-Amp medium and then sequence to further verify the inserted fragment. When the sequencing result is the target inserted fragment sequence, the clone of this colony is a positive clone; 120 positive clones are obtained. After culturing the 120 clones in LB-Amp medium respectively, add glycerol (final concentration 20%) and store at -80 °C; for subsequent plasmid extraction.
[0042] III. Preparation of the mixed standard plasmid
[0043] (1) Extraction of the plasmid carrying the target gene fragment
[0044] After culturing the above 120 positive clones overnight at 37 °C in LB-Amp medium, use the EZ Spin Column Plasmid DNA Preparation Kit to extract the plasmid. The specific steps are as follows:
[0045] ① Add 50 ml of the overnight culture to an appropriate centrifuge tube and centrifuge at a speed of 5000 rpm for 10 min. Discard the fermentation broth.
[0046] ② Add 5 ml of Solution I to the precipitate, gently mix, and let it stand on ice for 2 min.
[0047] ③ Add 5 ml of Solution II to the mixture, gently mix by inverting the tube 4 - 6 times, and then let it stand at room temperature for 2 min.
[0048] ④ Add 7 ml of Solution III and mix gently. Incubate at room temperature for 2 min.
[0049] ⑤ Centrifuge at 10,000 rpm for 10 min.
[0050] ⑥ Place the column into a 50 ml collection tube. Transfer the above supernatant (Step ⑤) to the column, let it stand at room temperature for 5 min. Centrifuge at 6,000 rpm for 5 min.
[0051] ⑦ Discard the effluent in the tube. Add 5 ml of washing solution to the column and centrifuge at 6,000 rpm for 5 min.
[0052] ⑧ Repeat the washing process in Step ⑦.
[0053] ⑨ Discard the effluent in the collection tube. Centrifuge additionally at 6,000 rpm for 5 min to remove the residual washing solution.
[0054] ⑩ Transfer the column to a clean 50 ml microcentrifuge tube. Add 500 μl of elution buffer to the central part of the column membrane, incubate at 37 °C for 2 min. Centrifuge at 6,000 rpm for 2 min. Store the plasmid DNA at -20 °C.
[0055] Quality inspection of the extracted plasmid: Verification of sequence accuracy, appearance inspection, OD ratio, restriction enzyme identification, presence or absence of RNA residue, and genomic residue.
[0056] Concentration determination and lyophilization: Use the Shandong Bieke BK - FD10S lyophilizer to lyophilize the plasmid to obtain plasmid dry powder. Finally, each tube contains 25 ng of each plasmid. Store the lyophilized product at -20 °C.
[0057] (2) Preparation of the mixed standard plasmid
[0058] Centrifuge the plasmid dry powder. First, dissolve the plasmid dry powder in the first tube with a certain volume of sterile TE buffer, and then use this plasmid solution to dissolve the plasmid dry powder in the next tube in turn. This process is repeated multiple times, using 500 μL of TE buffer each time until all are dissolved to prepare a 4200 μL mixed standard plasmid mother solution. The copy number of each target gene fragment in the mother solution is 2.3×10 9 ~2.8×10 9 copies / μL. After aliquoting, store the mixed standard plasmid mother solution at -20 °C for subsequent sensitivity verification.
[0059] IV. Sensitivity verification:
[0060] (1) Dilution of the mixed standard plasmid:
[0061] The mixed standard plasmid prepared above was diluted 10, 20, 100, 200, 1000, 2000, 10 4 、10 5 、10 6 、10 7 、10 8 times, etc. to obtain a series of concentrations of the mixed standard plasmid for subsequent ordinary qPCR and HT-qPCR detections. The gene copy number and DNA concentration after dilution are shown in Table 3.
[0062] (2) Verification of the sensitivity of the mixed standard plasmid by ordinary qPCR and HT-qPCR
[0063] Ordinary qPCR: A 20 μL reaction system was configured for ordinary qPCR. The system was 10 μL of 1×SYBR Green I Master Mix, 0.5 μM forward primer, 0.5 μM reverse primer, and nuclease-free sterile water. 2 μL of the mixed standard plasmid with different concentrations diluted in (1) was added as the template respectively, and nuclease-free sterile water was added to the NTC control instead of the template DNA. The amplification program was: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 30 s, annealing and extension at 60°C for 30 s, for 40 cycles, and finally heating from 60°C to 97°C at a heating rate of 4°C / s, and the melting curve was measured at 0.4°C as a measurement point. The instrument was 480.
[0064] The results of the ordinary qPCR of the mixed standard plasmid were as Figure 1 . The results showed that when the log value of the gene copy number was 2.4, a significant difference began to appear in the Ct value compared with that of the NTC; while when the log values were 8.1 and 8.4, there was no significant difference in the Ct value compared with that at the log value of 7.4; Based on this, the standard curve calculation of the mixed standard plasmid during ordinary qPCR is carried out within the range of log values from 2.4 to 7.4.
[0065] HT-qPCR: On the basis of ordinary qPCR, the mixed plasmids with each gradient diluted in (1) were used as the template DNA for HT-qPCR.
[0066] ① Preparation of enzyme - primer / sample mixing plates: According to the layout instructions of the instrument with 120 Assays * 42 Samples, prepare 384 - well "enzyme - primer mixing plates" and "enzyme - sample mixing plates" respectively. Specifically: First, mix 911 μL of 2×SYBR Green I Mix enzyme and 547 μL of nuclease - free sterile water, and then add the mixed solution to 120 wells in the 384 - well plate using a multi - channel pipette, 10.9 μL per well. Then, add 2.7 μL of primer solutions (10 μM) of 120 primer pairs to the 120 wells respectively. Thus, 120 groups of "enzyme - primer mixing plates" are obtained; First, mix 493 μL of 2×SYBR Green I Mix enzyme and 296 μL of nuclease - free sterile water, and then add the mixed solution to 42 wells in the 384 - well plate using a multi - channel pipette, 15.2 μL per well. Then, add 3.8 μL of mixed standard plasmid templates with different gradients to the 42 wells respectively. For the NTC control, add nuclease - free sterile water instead of template DNA. Thus, 42 groups of "enzyme - sample mixing plates" are obtained.
[0067] ② HT - qPCR sample addition: Use the SmartChip MyDesign high - throughput sample addition platform to add the reaction solutions in the 384 - well "enzyme - primer mixing plates" and "enzyme - sample mixing plates" to 5040 reaction wells on the SmartChip chip (a total of 5182 wells, and this layout occupies 5040 wells). Finally, the reaction system in each reaction well on the chip is 100 nL, containing 1×SYBRGreen I Master Mix, 0.5 μM forward primer, 0.5 μM reverse primer, template DNA, and nuclease - free sterile water.
[0068] ③ HT - qPCR: Perform fluorescence quantification using the SmartChip MyDesign high - throughput real - time quantitative PCR instrument (SmartChip Cycler). The amplification program is the same as that of ordinary qPCR.
[0069] The HT - qPCR results of the mixed standard plasmid are as Figure 2 . The results show that when the log value of the gene copy number is 3.4, a significant difference in Ct values begins to appear compared with the NTC; when the log values are 8.1 and 8.4, only the qPCR of some genes shows valid Ct values, but there is no significant difference in Ct values compared with those at the log value of 7.4. Based on this, the standard curve calculation of the mixed standard plasmid when using HT - qPCR is carried out in the range of log values from 3.4 to 7.4.
[0070] (3) Data analysis and verification of practical applications:
[0071] Calculation of the standard curve and amplification efficiency of the mixed standard plasmid: Linear regression analysis was performed using Excel 2019 (Microsoft, USA), with the logarithm of the standard plasmid copy number concentration on the abscissa and the Ct value on the ordinate.
[0072] The formula for calculating the qPCR amplification efficiency is as follows:
[0073] Eff = 10 (-1 / slope) -1
[0074] where slope refers to the slope of the standard curve. The statistical results of the amplification efficiency are as Figure 3 . During HT-qPCR, the amplification efficiency of 75% of the genes reached 0.9 or above, and this proportion was 77% in ordinary qPCR. However, the amplification efficiency of more than 98% of the genes was above 0.8 using both methods. The use of the standard mixed plasmid was verified in both methods.
[0075] Detection sensitivity of the mixed standard plasmid in different methods: The limits of quantification (LoQ) of ordinary qPCR and HT-qPCR for different genes are shown in Table 4. HT-qPCR has a smaller reaction system volume, so the LoQ is lower than that of ordinary qPCR, showing greater advantages than ordinary qPCR.
[0076] Comparison of the reliability of HT-qPCR and ordinary qPCR in the detection of the same batch of samples: We used HT-qPCR and ordinary qPCR to detect the same batch of samples to evaluate the reliability of the two methods in quantitative analysis. We selected multiple environmental samples and simultaneously performed HT-qPCR and ordinary qPCR quantitative detection on the 16S rRNA gene that exists in all environments. The specific method is shown in (2), where the template was changed from the mixed standard plasmid to environmental sample DNA with a concentration of 20 ng / μL. For the 26 samples detected, the ratio of the results of ordinary qPCR to HT-qPCR and its reciprocal are shown in Figure 4 , 90% of the ratios were between 0.4 and 1.4, far lower than 10. The difference in the gene copy numbers detected by the two methods for the same sample was far lower than one order of magnitude, further proving the accuracy, stability, and applicability of this mixed standard governance.
[0077] Table 1 Target fragment sequence information
[0078]
[0079]
[0080]
[0081]
[0082]
[0083]
[0084]
[0085]
[0086]
[0087] Table 2 Plasmid Verification and Quality Control
[0088] Quality control indicators Judgment criteria Results Accuracy of the sequence Alignment with the target sequence after Sanger sequencing Consistent Appearance inspection Transparent, colorless, without impurities or precipitation Meet OD: 260 / 280 1.8～2.0 Pass OD: 260 / 230 ≥2.0 Pass Restriction enzyme identification Agarose gel electrophoresis detects the expected size Pass RNA residue Invisible on agarose gel electrophoresis Not seen Genomic residue Invisible on agarose gel electrophoresis Not seen
[0089] Table 3 DNA Concentrations and Copy Numbers of Target Genes Corresponding to Different Gradient Mixed Standard Plasmids
[0090]
[0091] Table 4 Quantitative Limits of Detection (LoQ) for Different Genes by Conventional qPCR and HT-qPCR
[0092]
[0093]
[0094]
[0095]
[0096]
Claims
1. A method for preparing a hybrid standard plasmid, characterized in that, The steps are as follows: 1) Synthesize the target gene fragments with sequence numbers as shown in SEQ ID NO: 1 to 120 and the pESI-T vector to obtain 120 synthetic plasmids, and insert the target gene fragments into the EcoRI site of the pESI-T vector respectively; 2) Dissolve the synthetic plasmids with TE buffer to a final concentration of 25 ng / μL and then transform Escherichia coli competent cells, and introduce the synthetic plasmids into the cells to obtain transformation products; 3) Uniformly coat the transformation products on LB plates containing corresponding antibiotics; screen positive clones to obtain 120 positive clones; 4) Amplify and culture the 120 positive clones, and extract plasmids from the cultures; 5) Verify the plasmids; 6) Measure the plasmid concentration and prepare diluted standard plasmids.
2. The preparation method of the mixed standard plasmid according to claim 1, characterized in that The method for extracting plasmids from the cultures is to use an EZ Spin Column Plasmid DNA Preparation Kit for Drugs to extract plasmids.
3. The method for preparing the hybrid standard plasmid according to claim 2, wherein, The specific steps for introducing the synthetic plasmids into the cells are as follows: Take competent cells, dilute and aspirate the synthetic plasmids, add them to the competent cells, and mix well in a centrifuge tube; then place the centrifuge tube in an environment of 0 °C; partially immerse the centrifuge tube in a water bath for heat shock; After the heat shock ends, cool down the centrifuge tube; add culture medium for recovery culture; after the recovery ends, obtain transformation products.
4. The method for preparing a mixed standard plasmid according to claim 1, wherein, The method for extracting plasmids is as follows: Add the overnight culture to a centrifuge tube and centrifuge; completely drain the liquid; add 5 ml of Solution I to the precipitate, gently mix, and let it stand on ice. Add 5 ml of Solution II to the mixture, gently mix by inverting the tube 4 - 6 times, and then let it stand at room temperature; add 7 ml of Solution III and gently mix; incubate at room temperature; centrifuge; place the column into a collection tube; transfer the above supernatant to the column and let it stand at room temperature for 5 min. Centrifuge at 6000 rpm for 3 - 5 min; discard the effluent in the tube. Add a washing solution to the column and centrifuge for washing; Repeat the washing process; Discard the effluent in the collection tube; centrifuge for 5 min to remove the residual washing solution; transfer the column to a clean 50 ml microcentrifuge tube; add 500 μl of elution buffer to the central part of the column membrane and incubate for 2 min; centrifuge for 2 min.
5. The preparation method of the mixed standard plasmid according to claim 4, characterized in that, The mixed standard plasmid was prepared as follows: The plasmid dry powder was centrifuged and 4200 μL of TE buffer was added. The total concentration was 714.3 ng / μL, and the concentration of each plasmid was 5.95 ng / μL, so that the copy number of each target gene fragment was 10 9 copies / μL.
6. The method for preparing a mixed standard plasmid according to claim 5, wherein, The preparation method of the diluted standard plasmid is as follows: The prepared mixed standard plasmid is serially diluted 10-fold to obtain 10 8 ~10 copies / μL, and then 10 8 , 10 7 , 10 6 copies / μL are serially diluted two-fold to obtain plasmids with diluted concentrations.
7. A hybrid standard plasmid, characterized in that, It includes the gene sequences of SEQ ID NO: 1 - 120.
8. A kit, characterized in that, It includes the mixed standard plasmid described in claim 6.
9. Use of the mixed standard plasmid according to any one of claims 1 - 6 in HT-qPCR detection.