Universal method capable of preparing molecular weight internal standard and fluorescence spectrum correction reagent

Abstract

The invention relates to the technical field of biology, in particular to a universal method for preparing a molecular weight interior label and a fluorescence spectrum correction reagent. The invention provides a general preparation method of the molecular weight internal standard and the spectrum correction reagent based on the random sequence, the spectrum correction reagent and the molecular weight internal standard reagent can be produced at the same time, the production period is shortened, various spectrum correction reagents and molecular weight internal standards with various fragment sizes are adapted, and due to the fact that the same template raw materials are used, the production cost can be reduced; and the template sequence is a random sequence and has no homologous sequence with the human gene locus, so that laboratory pollution caused by template or PCR product leakage in the use process is avoided.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a universal method for preparing molecular weight internal standards and spectrum correction reagents. Background Art

[0002] Individual identification of DNA is mainly detected using a gene analyzer. The gene analyzer uses capillary electrophoresis technology to separate the fragments from small to large by taking advantage of the different molecular weights of DNA fragments. The laser excites the fluorescent dye carried by the DNA fragments, and the CCD element of the instrument converts the optical signal into an electrical signal, and the DNA fragments are displayed on the software in the form of a peak graph. The normal use of the instrument is inseparable from the spectral correction reagent and the molecular weight internal standard. The spectral correction reagent contains all the fluorescent dyes used by the reagents to be detected. By spectrally correcting the instrument, the instrument can recognize fluorescence without causing fluorescence penetration. The role of the molecular weight internal standard is equivalent to the marker in gel electrophoresis. It is composed of a series of DNA fragments of known fragment sizes and is used as a ruler to measure the molecular weight of the DNA fragment to be tested.

[0003] The spectral correction reagent mainly utilizes the PCR method. By designing primers of different fragment sizes, the spectral primers amplify the corresponding templates to obtain DNA fragments with different fluorescent labels and fragment sizes. The ratio is adjusted for mixing so that the peak heights of each fragment displayed on the instrument are not much different.

[0004] There are two main methods for preparing molecular weight internal standards. One is to design a fixed upstream fluorescent labeled primer on the vector plasmid through PCR technology, and then design downstream non-labeled primers of different fragment sizes, obtain DNA fragments of different fragment sizes through amplification, adjust the ratio and mix them so that the peak heights of each fragment displayed on the instrument are not much different. The other is site-directed mutagenesis and enzyme digestion technology. After cloning the target DNA fragment into the vector plasmid, site-directed mutagenesis is performed at different lengths of the target DNA fragment to introduce restriction enzyme cutting sites, and then amplification is performed using fluorescently labeled primers to obtain PCR products, and restriction endonucleases are used to digest the PCR products to obtain DNA amplification products of different sizes.

[0005] The existing preparation methods of molecular weight internal standards and spectral correction reagents have defects as follows.

[0006] (I) Long preparation cycle: In the existing technical solutions, the preparation methods of spectral correction reagents and molecular weight internal standards are not universal, and the two reagents need to be prepared in batches.

[0007] (ii) High cost: The amplification of the spectral calibration reagent requires the use of a DNA template with corresponding primers. For example, six DNA templates may be used to make a six-color spectral calibration reagent.

[0008] (III) There is a risk of contamination: If the template used in the production of spectral calibration reagents or molecular weight internal standards is human DNA or DNA from other species, it may cause contamination to other experiments using DNA from the same species. Summary of the invention

[0009] In view of this, the present invention provides a general method for preparing molecular weight internal standards and spectral calibration reagents. The template sequence is an artificially synthesized random sequence, which has no strict requirements and is easy to obtain; the template sequence has no homologous sequence with the human genome and will not cause contamination to other reagents during use; the method has wide applicability and is compatible with the preparation of molecular weight internal standards of various molecular weights and spectral calibration reagents of various color combinations. In short, by using an artificially synthesized random sequence as an amplification template, the present invention overcomes the problems of long production cycle, high cost, and DNA contamination risk in the prior art.

[0010] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions.

[0011] The invention provides a universal preparation method of a molecular weight internal standard and a spectrum correction reagent. The molecular weight internal standard and the spectrum correction reagent are both prepared based on the same template, and the sequence of the template is a random sequence.

[0012] In some specific embodiments of the present invention, the above general preparation method comprises the following steps:

[0013] S1-1. Design and synthesize a primer set according to the size of each fragment of the product and the sequence of the template, wherein the upstream primer of the primer set has a fluorescent label;

[0014] S1-2, amplifying the template using the primer set to obtain an amplified product;

[0015] S1-3, diluting the amplified product to obtain a sample;

[0016] S1-4, mixing formamide with the sample, performing electrophoresis, and obtaining an electrophoresis result;

[0017] S1-5, adjusting the concentration of the sample according to the electrophoresis result to obtain the product;

[0018] The sequence of the template is a random sequence;

[0019] The product is a molecular weight internal standard or a spectrum calibration reagent.

[0020] In some specific embodiments of the present invention, the random sequence described in the above general preparation method has no homologous part with the human genome.

[0021] In some specific embodiments of the present invention, the length of the template in the above general preparation method is 1000 bp.

[0022] In some specific embodiments of the present invention, the fluorescent label in the above general preparation method is one of FAM, HEX, TAMRA, ROX, LPUR, LIZ, or a combination of two or more thereof.

[0023] In some specific embodiments of the present invention, the mixing ratio of the formamide and the sample in the above general preparation method is 10:0.2 or 10:0.3.

[0024] In some specific embodiments of the present invention, the above general preparation method comprises the following steps:

[0025] S2-1, designing and synthesizing a molecular weight internal standard primer set according to the size of each molecular weight internal standard fragment and the sequence of the template, wherein the upstream primer of the molecular weight internal standard primer set has a fluorescent label;

[0026] S2-2, amplifying the template using the molecular weight internal standard primer set to obtain a molecular weight internal standard amplification product;

[0027] S2-3, diluting the molecular weight internal standard amplification product 10 times to obtain a molecular weight internal standard amplification product dilution solution, taking 2 μL of the molecular weight internal standard amplification product dilution solution and mixing it with nuclease-free water to obtain 500 μL of a molecular weight internal standard sample;

[0028] S2-4, mixing formamide and the molecular weight internal standard sample in a ratio of 10:0.2, and performing electrophoresis to obtain electrophoresis result 1;

[0029] S2-5, adjusting the concentration of the molecular weight internal standard sample according to the electrophoresis result 1 to obtain a molecular weight internal standard;

[0030] S2-6. Designing and synthesizing a spectral correction reagent primer set according to the sizes of each fragment of the spectral correction reagent and the sequence of the template, wherein the upstream primer of the spectral correction reagent primer set has a fluorescent label;

[0031] S2-7, amplifying the template using the spectral correction reagent primer set to obtain a spectral correction reagent amplification product;

[0032] S2-8, dilute the spectral correction reagent amplification product 10 times to obtain a spectral correction reagent amplification product dilution solution, take 2 μL of the spectral correction reagent amplification product dilution solution and mix it with nuclease-free water to obtain 500 μL of spectral correction reagent sample;

[0033] S2-9, mixing formamide and the spectrum correction reagent sample in a ratio of 10:0.3, and performing electrophoresis to obtain electrophoresis result 2;

[0034] S2-10, adjusting the sample concentration of the spectrum correction reagent according to the electrophoresis result 2 to obtain a spectrum correction reagent;

[0035] The sequence of the template is a random sequence;

[0036] The fluorescent label is one of FAM, HEX, TAMRA, ROX, LPUR, and LIZ, or a combination of two or more thereof.

[0037] The present invention also provides a reagent combination, including a molecular weight internal standard and a spectrum correction reagent;

[0038] The molecular weight internal standard includes molecular weight internal standard NS500 and / or molecular weight internal standard NS600;

[0039] The molecular weight internal standard NS500 includes a primer having a sequence as shown in SEQ ID NO: 1, a primer having a sequence as shown in SEQ ID NO: 2, a primer having a sequence as shown in SEQ ID NO: 3, a primer having a sequence as shown in SEQ ID NO: 4, a primer having a sequence as shown in SEQ ID NO: 5, a primer having a sequence as shown in SEQ ID NO: 6, a primer having a sequence as shown in SEQ ID NO: 7, a primer having a sequence as shown in SEQ ID NO: 8, a primer having a sequence as shown in SEQ ID NO: 9, a primer having a sequence as shown in SEQ ID NO: 10, a primer having a sequence as shown in SEQ ID NO: 11, a primer having a sequence as shown in SEQ ID NO: 12, a primer having a sequence as shown in SEQ ID NO: 13, a primer having a sequence as shown in SEQ ID NO: 14, and a primer having a sequence as shown in SEQ ID NO: 15;

[0040] The molecular weight internal standard NS600 includes a primer with a sequence as shown in SEQ ID NO: 1, a primer with a sequence as shown in SEQ ID NO: 3, a primer with a sequence as shown in SEQ ID NO: 6, a primer with a sequence as shown in SEQ ID NO: 7, a primer with a sequence as shown in SEQ ID NO: 9, a primer with a sequence as shown in SEQ ID NO: 10, a primer with a sequence as shown in SEQ ID NO: 12, a primer with a sequence as shown in SEQ ID NO: 15, a primer with a sequence as shown in SEQ ID NO: 16, a primer with a sequence as shown in SEQ ID NO: 17, a primer with a sequence as shown in SEQ ID NO: 18, a primer with a sequence as shown in SEQ ID NO: 19, a primer with a sequence as shown in SEQ ID NO: 20, a primer with a sequence as shown in SEQ ID NO: 21, a primer with a sequence as shown in SEQ ID NO: 22, a primer with a sequence as shown in SEQ ID NO: 23, a primer with a sequence as shown in SEQ ID NO: 24, a primer with a sequence as shown in SEQ ID NO: 25, a primer with a sequence as shown in SEQ ID NO: The primers shown in SEQ ID NO: 26, the primers shown in SEQ ID NO: 27, the primers shown in SEQ ID NO: 28, the primers shown in SEQ ID NO: 29, the primers shown in SEQ ID NO: 30, the primers shown in SEQ ID NO: 31, the primers shown in SEQ ID NO: 32, the primers shown in SEQ ID NO: 33, the primers shown in SEQ ID NO: 34, the primers shown in SEQ ID NO: 35, and the primers shown in SEQ ID NO: 36;

[0041] The spectrum correction reagent includes a five-color spectrum correction reagent and / or a six-color spectrum correction reagent;

[0042] The five-color spectrum correction reagent includes a primer with a LIZ-labeled sequence as shown in SEQ ID NO: 1, a primer with a label as shown in SEQ ID NO: 3, a primer with a FAM sequence as shown in SEQ ID NO: 37, a primer with a HEX sequence as shown in SEQ ID NO: 38, a primer with a TAMRA sequence as shown in SEQ ID NO: 39, and a primer with a ROX sequence as shown in SEQ ID NO: 40;

[0043] The six-color spectral correction reagent includes a primer with a LIZ-labeled sequence as shown in SEQ ID NO: 1, a primer with a labeled sequence as shown in SEQ ID NO: 3, a primer with a FAM sequence as shown in SEQ ID NO: 37, a primer with a HEX sequence as shown in SEQ ID NO: 38, a primer with a TAMRA sequence as shown in SEQ ID NO: 39, a primer with a ROX sequence as shown in SEQ ID NO: 40, and a primer with a LPUR-labeled sequence as shown in SEQ ID NO: 41.

[0044] The effects of the present invention are as follows.

[0045] (I) Universality. Spectral calibration reagents and molecular weight internal standard reagents can be produced at the same time, shortening the production cycle; the method is suitable for various spectral calibration reagents (common 5-color, 6-color, and 8-color spectra) and molecular weight internal standards of various fragment sizes.

[0046] (ii) Low cost. Because a universal production method is used, the spectral correction reagent and the molecular weight internal standard reagent use the same template raw material, which can reduce the production cost.

[0047] (III) No risk of contamination. The template sequence is a random sequence. After NCBI BLAST comparison, there is no homologous sequence with the human locus. There will be no laboratory contamination due to leakage of template or PCR product during use. BRIEF DESCRIPTION OF THE DRAWINGS

[0048] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art are briefly introduced below.

[0049] Figure 1 Shows the test results of the molecular weight internal standard NS500;

[0050] Figure 2 Shows the test results of the molecular weight internal standard NS600;

[0051] Figure 3 Shows the test results of the five-color spectrum calibration reagent;

[0052] Figure 4 Shows the detection results of the six-color spectral calibration reagent. DETAILED DESCRIPTION

[0053] The present invention discloses a general method for preparing molecular weight internal standards and spectral correction reagents. Those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It is particularly important to point out that all similar substitutions and modifications are obvious to those skilled in the art, and they are all considered to be included in the present invention. The methods and applications of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the methods and applications described herein without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0054] The specific embodiments of the present invention are as follows.

[0055] 1. Randomly generate template sequences and compare them

[0056] Use software / webpage with relevant functions to randomly generate a DNA sequence of 1000 bp in length. After the sequence is generated, it is compared on blastn to ensure that the random sequence has no homologous parts with the human genome.

[0057] 2. Use the random sequence as a template to design primers

[0058] Molecular weight internal standard: Using the random sequence as a template, design an upstream primer fixed as a molecular weight internal standard fluorescent label primer. Fluorescent labels include but are not limited to FAM, HEX, TAMRA, ROX, LPUR, LIZ, etc. Design a downstream primer based on the required fragment size;

[0059] Spectral correction reagent: Using the random sequence as a template, design upstream and downstream primers according to the required spectral reagents. Design 5 pairs of primers for 5-color spectral reagents, design 6 pairs of primers for 6-color spectral reagents, and so on. Fluorescent labels include but are not limited to FAM, HEX, TAMRA, ROX, LPUR, LIZ, etc.

[0060] 3. Synthesize template sequence plasmid and primers

[0061] The template plasmid (vectors including but not limited to pUC57, pUC19, etc.) and primers are synthesized by an external biological company.

[0062] 4. PCR Amplification

[0063] The amplification system was constructed as shown in Table 1.

[0064] Table 1

[0065]

[0066] PCR amplification conditions: 95°C, 1 min; (95°C, 10 s, 60°C, 1 min) × 40 cycles; 60°C, 60 min; 4°C, hold.

[0067] 5. Sample calibration

[0068] Molecular weight internal standard reagent:

[0069] (1) Dilute 10 μL of PCR product 10 times to obtain 100 μL of PCR product dilution solution;

[0070] (2) Take 2 μL of each PCR product dilution and mix them, then add nuclease-free water to 500 μL to obtain a molecular weight internal standard sample;

[0071] (3) Electrophoresis detection: Mix formamide and molecular weight internal standard sample at a ratio of 10:0.2 (v:v) and dispense 10 μL into each reaction well.

[0072] Spectral calibration reagents:

[0073] (1) Dilute 10 μL of PCR product 10 times to obtain 100 μL of PCR product dilution solution;

[0074] (2) Take 2 μL of each PCR product dilution solution, mix them, and add nuclease-free water to 500 μL to obtain a spectral calibration reagent sample;

[0075] (3) Electrophoresis detection: Mix formamide and spectral calibration reagent sample at a ratio of 10:0.3 (v:v) and dispense 10 μL into each reaction well.

[0076] 6. Level the product according to the recipe

[0077] According to the peak height results of electrophoresis detection, the volume of PCR product diluent is increased or decreased to achieve the effect of adjusting the peak height.

[0078] Some concepts involved in the present invention are as follows.

[0079] Spectral calibration reagent: Spectral calibration reagent is a chemical substance or mixture used to adjust and optimize the performance of instruments during spectral analysis. By using spectral calibration reagents, the wavelength accuracy, intensity linearity and stability of the instrument can be corrected, thereby improving the quality of analytical data. For genetic analyzers, spectral calibration reagents can help genetic analyzers identify fluorescence and reduce the penetration between fluorescence.

[0080] Molecular weight internal standard: Molecular weight internal standard refers to a standard substance of known molecular weight added to the sample during mass analysis or molecular weight determination. Its main function is to serve as a reference to help correct and standardize changes in experimental conditions and improve the accuracy and reproducibility of test results. By comparing the internal standard with the sample, the molecular weight or concentration of the sample can be accurately determined.

[0081] Unless otherwise specified, the raw materials, reagents, consumables and instruments involved in the present invention are all common commercial products and can be purchased from the market.

[0082] The present invention will be further described below in conjunction with the embodiments.

[0083] Example 1: Obtaining a universal plasmid

[0084] 1. Use a website with a random sequence generation function (Random DNA Sequence) to randomly generate a DNA sequence with a fragment length of 1000 bp. The sequence is as follows:

[0085]

[0086] 2. Compare the sequence on the NCBI website and confirm that the sequence has no homologous sequence with the human genome.

[0087] 3. The sequence plasmid was sent to an external gene synthesis manufacturer (Shengong / Qingke) for synthesis. The vector was selected as pUC57, and the plasmid mass was 4 μg.

[0088] Example 2: Preparation of molecular weight internal standard NS500

[0089] 1. Primers were designed according to the plasmid template sequence in Example 1, with a primer Tm value of 58-64°C, and molecular weight internal standard fragment sizes including: 75 bp, 100 bp, 139 bp, 150 bp, 160 bp, 200 bp, 250 bp, 300 bp, 340 bp, 350bp, 400 bp, 450 bp, 490 bp, and 500 bp.

[0090] 2. All molecular weight internal standard fragments share the same fluorescent primer sequence, and fluorescent markers include but are not limited to: FAM, HEX, TAMRA, ROX, LPUR, LIZ, NH650A. The fluorescent marker used in this embodiment is LIZ.

[0091] 3. The primers were synthesized by a gene synthesis company. The specific primer sequences are shown in Table 2.

[0092] Table 2

[0093]

[0094] 4. Use 1× Low TE solution (Biogen) to dissolve the primer powder according to the indicated concentration and dilute to 10 μM.

[0095] 5. Use 1× Low TE solution (Sangong) to dissolve and dilute the plasmid powder (4 μg) in Example 1 to a final concentration of 0.04 ng / μL.

[0096] 6. Prepare the amplification reaction system according to Table 3

[0097] Table 3

[0098]

[0099] 7. Amplification conditions

[0100] Amplification was performed using a 9700 PCR instrument (ABI), and the amplification conditions are shown in Table 4.

[0101] Table 4

[0102]

[0103] 8. Sample calibration

[0104] After diluting 10 μL of PCR product 10 times, 100 μL of PCR product dilution solution was obtained. 2 μL of PCR product dilution solution was taken and mixed, and nuclease-free water was added to 500 μL to obtain the molecular weight internal standard sample.

[0105] Electrophoresis detection: Mix formamide and molecular weight internal standard sample at a ratio of 10:0.2, and dispense 10 μL into each reaction well. Detection was performed using 3500xL DX Genetic Analyzer (ABI).

[0106] 9. Adjust the internal standard according to the ratio

[0107] According to the peak height results of electrophoresis detection, increase or decrease the volume of PCR product diluent to adjust the peak height so that the ratio of the lowest fragment peak height to the highest fragment peak height is ≥ 0.5. Perform electrophoresis detection again. The test results are as follows Figure 1 shown.

[0108] Example 3: Preparation of molecular weight internal standard NS600

[0109] 1. Primers were designed according to the plasmid template sequence in Example 1, with a primer Tm value of 58-64°C, and molecular weight internal standard fragment sizes including: 60 bp, 80 bp, 100 bp, 120 bp, 140 bp, 160 bp, 180 bp, 200 bp, 220 bp, 240bp, 260 bp, 280 bp, 300 bp, 314 bp, 320 bp, 340 bp, 360 bp, 380 bp, 400 bp, 420 bp, 440bp, 460 bp, 480 bp, 500 bp, 525 bp, 550 bp, 575 bp, and 600 bp.

[0110] 2. All molecular weight internal standard fragments share the same fluorescent primer sequence, and fluorescent markers include but are not limited to: FAM, HEX, TAMRA, ROX, LPUR, LIZ, NH650A. The fluorescent marker used in this embodiment is LIZ.

[0111] 3. The primers were synthesized by a gene synthesis company. The specific primer sequences are shown in Table 5.

[0112] Table 5

[0113]

[0114] 4. Use 1× Low TE solution (Biogen) to dissolve the primer powder according to the indicated concentration and dilute to 10 μM.

[0115] 5. Use 1× Low TE solution (Sangong) to dissolve and dilute the plasmid powder (4 μg) in Example 1 to a final concentration of 0.04 ng / μL.

[0116] 6. Prepare the amplification reaction system according to Table 6

[0117] Table 6

[0118]

[0119] 7. Amplification conditions

[0120] Amplification was performed using a 9700 PCR instrument (ABI), and the amplification conditions are shown in Table 7.

[0121] Table 7

[0122]

[0123] 8. Sample calibration

[0124] After diluting 10 μL of PCR product 10 times, 100 μL of PCR product dilution solution was obtained. 2 μL of PCR product dilution solution was taken and mixed, and nuclease-free water was added to 500 μL to obtain the molecular weight internal standard sample.

[0125] Electrophoresis detection: Mix formamide and molecular weight internal standard sample at a ratio of 10:0.2, and dispense 10 μL into each reaction well. Detection was performed using 3500xL DX Genetic Analyzer (ABI).

[0126] 9. Adjust the internal standard according to the ratio

[0127] According to the peak height results of electrophoresis detection, increase or decrease the volume of PCR product diluent to adjust the peak height so that the ratio of the lowest fragment peak height to the highest fragment peak height is ≥ 0.5. Perform electrophoresis detection again. The test results are as follows Figure 2 shown.

[0128] Example 4: Five-color spectrum calibration reagent

[0129] 1. Design primers according to the plasmid template sequence in Example 1, with a primer Tm value of 58-64°C.

[0130] 2. All spectral calibration reagent fragments share the same non-labeled primer sequence, and fluorescent labels include but are not limited to: FAM, HEX, TAMRA, ROX, LPUR, LIZ, NH650A. The fluorescent labels used in this embodiment are FAM, HEX, TAMRA, ROX, and LIZ.

[0131] 3. The primers were synthesized by a gene synthesis company. The specific primer sequences are shown in Table 8.

[0132] Table 8

[0133]

[0134] 4. Use 1× Low TE solution (Biogen) to dissolve the primer powder according to the indicated concentration and dilute to 10 μM.

[0135] 5. Use 1× Low TE solution (Sangong) to dissolve and dilute the plasmid powder (4 μg) in Example 1 to a final concentration of 0.04 ng / μL.

[0136] 6. Prepare the amplification reaction system according to Table 9

[0137] Table 9

[0138]

[0139] 7. Amplification conditions

[0140] Amplification was performed using a 9700 PCR instrument (ABI), and the amplification conditions are shown in Table 10.

[0141] Table 10

[0142]

[0143] 8. Sample calibration

[0144] After diluting 10 μL of PCR product 10 times, 100 μL of PCR product dilution solution was obtained. 2 μL of PCR product dilution solution was taken and mixed, and nuclease-free water was added to 500 μL to obtain the spectral calibration reagent sample.

[0145] Electrophoresis detection: Mix formamide and spectral calibration reagent sample at a ratio of 10:0.3 and dispense 10 μL into each reaction well. Detection was performed using a 3500xL DX Genetic Analyzer (ABI).

[0146] 9. Adjust the internal standard according to the ratio

[0147] According to the peak height results of electrophoresis detection, increase or decrease the volume of PCR product diluent to adjust the peak height so that the ratio of the lowest fragment peak height to the highest fragment peak height is ≥ 0.5. Perform electrophoresis detection again. The test results are as follows Figure 3 shown.

[0148] Example 5: Six-color spectral calibration reagent

[0149] 1. Design primers according to the plasmid template sequence in Example 1, with a primer Tm value of 58-64°C.

[0150] 2. All spectral calibration reagent fragments share the same non-labeled primer sequence, and fluorescent labels include but are not limited to: FAM, HEX, TAMRA, ROX, LPUR, LIZ, NH650A. The fluorescent labels used in this embodiment are FAM, HEX, TAMRA, ROX, LPUR, and LIZ.

[0151] 3. The primers were synthesized by a gene synthesis company. The specific primer sequences are shown in Table 11.

[0152] Table 11

[0153]

[0154] 4. Use 1× Low TE solution (Biogen) to dissolve the primer powder according to the indicated concentration and dilute to 10 μM.

[0155] 5. Use 1× Low TE solution (Sangong) to dissolve and dilute the plasmid powder (4 μg) in Example 1 to a final concentration of 0.04 ng / μL.

[0156] 6. Prepare the amplification reaction system according to Table 12

[0157] Table 12

[0158]

[0159] 7. Amplification conditions

[0160] Amplification was performed using a 9700 PCR instrument (ABI), and the amplification conditions are shown in Table 13.

[0161] Table 13

[0162]

[0163] 8. Sample calibration

[0164] After diluting 10 μL of PCR product 10 times, 100 μL of PCR product dilution solution was obtained. 2 μL of PCR product dilution solution was taken and mixed, and nuclease-free water was added to 500 μL to obtain the spectral calibration reagent sample.

[0165] Electrophoresis detection: Mix formamide and spectral calibration reagent sample at a ratio of 10:0.3 and dispense 10 μL into each reaction well. Detection was performed using a 3500xL DX Genetic Analyzer (ABI).

[0166] 9. Adjust the internal standard according to the ratio

[0167] According to the peak height results of electrophoresis detection, increase or decrease the volume of PCR product diluent to adjust the peak height so that the ratio of the lowest fragment peak height to the highest fragment peak height is ≥ 0.5. Perform electrophoresis detection again. The test results are as follows Figure 4 shown.

[0168] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A general method for preparing a molecular weight internal standard and a spectral correction reagent, characterized in that: The molecular weight internal standard and the spectrum correction reagent are both prepared based on the same template, and the sequence of the template is a random sequence.

2. The general preparation method according to claim 1, characterized in that: The random sequence has no homologous part with the human genome.

3. The general preparation method according to claim 1 or 2, characterized in that: The length of the template is 1000 bp.

4. The general preparation method according to claim 1, characterized in that: The following steps are involved: S1-1. Design and synthesize a primer set according to the size of each fragment of the product and the sequence of the template, wherein the upstream primer of the primer set has a fluorescent label; S1-2, amplifying the template using the primer set to obtain an amplified product; S1-3, diluting the amplified product to obtain a sample; S1-4, mixing formamide with the sample, performing electrophoresis, and obtaining an electrophoresis result; S1-5, adjusting the concentration of the sample according to the electrophoresis result to obtain the product; The sequence of the template is a random sequence; The product is a molecular weight internal standard or a spectrum calibration reagent.

5. The general preparation method according to claim 4, characterized in that: The random sequence has no homologous part with the human genome.

6. The general preparation method according to claim 4 or 5, characterized in that: The length of the template is 1000 bp.

7. The general preparation method according to any one of claims 4 to 6, characterized in that: The fluorescent label is one of FAM, HEX, TAMRA, ROX, LPUR, and LIZ, or a combination of two or more thereof.

8. The general preparation method according to any one of claims 4 to 7, characterized in that: The mixing ratio of the formamide to the sample is 10:0.2 or 10:0.

3.

9. The general preparation method according to claim 1, characterized in that: The following steps are involved: S2-1, designing and synthesizing a molecular weight internal standard primer set according to the size of each molecular weight internal standard fragment and the sequence of the template, wherein the upstream primer of the molecular weight internal standard primer set has a fluorescent label; S2-2, amplifying the template using the molecular weight internal standard primer set to obtain a molecular weight internal standard amplification product; S2-3, diluting the molecular weight internal standard amplification product 10 times to obtain a molecular weight internal standard amplification product dilution solution, taking 2 μL of the molecular weight internal standard amplification product dilution solution and mixing it with nuclease-free water to obtain 500 μL of a molecular weight internal standard sample; S2-4, mixing formamide and the molecular weight internal standard sample in a ratio of 10:0.2, and performing electrophoresis to obtain electrophoresis result 1; S2-5, adjusting the concentration of the molecular weight internal standard sample according to the electrophoresis result 1 to obtain a molecular weight internal standard; S2-6. Designing and synthesizing a spectral correction reagent primer set according to the sizes of each fragment of the spectral correction reagent and the sequence of the template, wherein the upstream primer of the spectral correction reagent primer set has a fluorescent label; S2-7, amplifying the template using the spectral correction reagent primer set to obtain a spectral correction reagent amplification product; S2-8, diluting the spectral correction reagent amplification product by 10 times to obtain a spectral correction reagent amplification product dilution solution, taking 2 μL of the spectral correction reagent amplification product dilution solution and mixing it with nuclease-free water to obtain a 500 μL spectral correction reagent sample; S2-9, mixing formamide and the spectrum correction reagent sample in a ratio of 10:0.3, and performing electrophoresis to obtain electrophoresis result 2; S2-10, adjusting the sample concentration of the spectrum correction reagent according to the electrophoresis result 2 to obtain a spectrum correction reagent; The sequence of the template is a random sequence; The fluorescent label is one of FAM, HEX, TAMRA, ROX, LPUR, and LIZ, or a combination of two or more thereof.

10. A reagent combination, characterized in that Includes molecular weight internal standards and spectral calibration reagents; The molecular weight internal standard includes molecular weight internal standard NS500 and / or molecular weight internal standard NS600; The molecular weight internal standard NS500 includes a primer having a sequence as shown in SEQ ID NO: 1, a primer having a sequence as shown in SEQ ID NO: 2, a primer having a sequence as shown in SEQ ID NO: 3, a primer having a sequence as shown in SEQ ID NO: 4, a primer having a sequence as shown in SEQ ID NO: 5, a primer having a sequence as shown in SEQ ID NO: 6, a primer having a sequence as shown in SEQ ID NO: 7, a primer having a sequence as shown in SEQ ID NO: 8, a primer having a sequence as shown in SEQ ID NO: 9, a primer having a sequence as shown in SEQ ID NO: 10, a primer having a sequence as shown in SEQ ID NO: 11, a primer having a sequence as shown in SEQ ID NO: 12, a primer having a sequence as shown in SEQ ID NO: 13, a primer having a sequence as shown in SEQ ID NO: 14, and a primer having a sequence as shown in SEQ ID NO: 15; The molecular weight internal standard NS600 includes a primer having a sequence as shown in SEQ ID NO: 1, a primer having a sequence as shown in SEQ ID NO: 3, a primer having a sequence as shown in SEQ ID NO: 6, a primer having a sequence as shown in SEQ ID NO: 7, a primer having a sequence as shown in SEQ ID NO: 9, a primer having a sequence as shown in SEQ ID NO: 10, a primer having a sequence as shown in SEQ ID NO: 12, a primer having a sequence as shown in SEQ ID NO: 15, a primer having a sequence as shown in SEQ ID NO: 16, a primer having a sequence as shown in SEQ ID NO: 17, a primer having a sequence as shown in SEQ ID NO: 18, a primer having a sequence as shown in SEQ ID NO: 19, a primer having a sequence as shown in SEQ ID NO: 20, a primer having a sequence as shown in SEQ ID NO: 21, a primer having a sequence as shown in SEQ ID NO: 22, a primer having a sequence as shown in SEQ ID NO: 23, a primer having a sequence as shown in SEQ ID NO: 24, a primer having a sequence as shown in SEQ ID NO: 25, a primer having a sequence as shown in SEQ ID NO: The primers shown in SEQ ID NO: 26, the primers shown in SEQ ID NO: 27, the primers shown in SEQ ID NO: 28, the primers shown in SEQ ID NO: 29, the primers shown in SEQ ID NO: 30, the primers shown in SEQ ID NO: 31, the primers shown in SEQ ID NO: 32, the primers shown in SEQ ID NO: 33, the primers shown in SEQ ID NO: 34, the primers shown in SEQ ID NO: 35, and the primers shown in SEQ ID NO: 36; The spectrum correction reagent includes a five-color spectrum correction reagent and / or a six-color spectrum correction reagent; The five-color spectrum correction reagent includes a primer with a LIZ-labeled sequence as shown in SEQ ID NO: 1, a primer with a label as shown in SEQ ID NO: 3, a primer with a FAM sequence as shown in SEQ ID NO: 37, a primer with a HEX sequence as shown in SEQ ID NO: 38, a primer with a TAMRA sequence as shown in SEQ ID NO: 39, and a primer with a ROX sequence as shown in SEQ ID NO: 40; The six-color spectral correction reagent includes a primer with a LIZ-labeled sequence as shown in SEQ ID NO: 1, a primer with a label as shown in SEQ ID NO: 3, a primer with a FAM sequence as shown in SEQ ID NO: 37, a primer with a HEX sequence as shown in SEQ ID NO: 38, a primer with a TAMRA sequence as shown in SEQ ID NO: 39, a primer with a ROX sequence as shown in SEQ ID NO: 40, and a primer with a LPUR-labeled sequence as shown in SEQ ID NO: 41.

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