CN108085367A - A kind of genetic analyzer tests special allele standard control preparation method of reagent thereof - Google Patents

Abstract

The invention discloses a method for preparing a special allele standard control reagent for testing a genetic analyzer, comprising: 1) obtaining a specific number of biological samples; 2) obtaining a series of alleles at a designated site by PCR; 3) a core sequence Site-directed mutagenesis; 4) Obtain all gene cloning vectors at designated sites; 5) Design and synthesize fluorescent primers; 6) Explore the concentration conditions of allelic cloning vectors and perform leveling tests; 6) Multi-template fluorescent multiplex amplification and detection; 7 ) Allelic standard substance amplification preparation, purification, preservation. The present invention adopts cloning technology to clone the sequence alleles of specific STR site series into selected cloning vectors, and then performs fluorescence amplification on the above-mentioned vectors through multi-template compound amplification technology, so as to realize the sustainability of allelic standard control reagents preparation.

Description

Preparation method of special allele standard control reagent for genetic analyzer test

technical field

The invention relates to the technical field of DNA testing, in particular to a method for preparing a special allele standard control reagent for genetic analyzer testing.

Background technique

DNA testing technology, especially DNA testing technology based on STR-PCR, has been widely used in individual identification and kinship determination after years of development. Based on the unique technical advantages of DNA testing technology, it has become a sharp sword for my country's public security organs to investigate and solve crimes, and it is the undisputed king of evidence in the field of forensic science. At the same time, it is also widely used in individual identification in civil cases, large-scale catastrophic accidents and mass emergencies.

In recent years, the distinction and identification of different individuals based on polymorphic short tandem repeat sequences has become the mainstream DNA typing technology for forensic DNA detection and individual identification. In particular, the recently developed multi-site fluorescent STR composite amplification technology has the advantages of high efficiency, rapidity, and high sensitivity, and has played a very important role in personal identification and paternity testing. However, the application of this technology must be effectively supported by the corresponding testing equipment platform and testing reagents.

my country's forensic DNA analysis technology started earlier. As early as 1985, when DNA fingerprint technology was first reported in the world for forensic identification, Chinese forensic scientists have been committed to the research of DNA testing technology. Obtained important results and realized localization. However, it has always relied on imports in terms of inspection equipment platforms, forming a high dependence on foreign products. Due to the above situation, my country initiated the development of a multi-channel capillary genetic analyzer based on laser-induced fluorescence detection technology during the "Eleventh Five-Year Plan" period. During the research period, a number of key technologies were overcome, and finally the localization of the instrument platform was realized, and it was quickly promoted and applied to DNA laboratories of many public security organs. The successful development of the domestic genetic analyzer is a major breakthrough and innovation in the field of forensic DNA testing technology in my country. It breaks the long-term technology and market monopoly of foreign countries in this field and meets the needs of my country's public security organs for DNA testing of criminal cases and database construction. , Play a very important role in promoting the advancement of public security technology and reducing the cost of DNA testing. With the rapid development of my country's forensic DNA testing technology, the localization process of genetic analyzers is accelerating, and the demand for domestic genetic analyzers in the field of forensic DNA testing is increasing. This is bound to affect the quality control and new models of domestic DNA testing equipment after mass production Equipment performance evaluation capabilities put forward higher requirements.

Genetic analyzer test standard control reagent is usually composed of a mixture of fluorescently labeled DNA of different lengths, and is one of the necessary reagents for verification, detection and testing of genetic analyzers. Standard reagents are very important for the quality control and performance testing of genetic analyzers, and are important media for evaluating and analyzing performance indicators such as typing accuracy, repeatability, screening accuracy, and sensitivity of genetic analyzers. By analyzing the results of electrophoresis separation of standard products on the genetic analyzer, the equipment's DNA screening ability, detection sensitivity, signal-to-noise ratio, test repeatability (between different groups and between different wells), fluorescence detection performance, etc. The indicators form systematic and objective evaluation results, so as to meet the requirements of quality control, performance testing, and new equipment development and testing of genetic analyzers. Currently, there is no commercial genetic analyzer test standard in the market. At present, commercial allele standards are usually used as test standard. There are a variety of commercial allelic ladders available in the market. However, the main genotyping standards mainly rely on imports from several large foreign companies. The price is expensive and the consumption of reagents is large, which undoubtedly greatly increases the production cost of the instrument, and only allelic genotyping standards are provided for imported equipment. The deployment of related laser excitation and signal acquisition conditions, the selection of fluorescence, and the concentration of standard substances cannot meet the testing requirements of domestic equipment platforms, which is not conducive to the establishment of equipment quality control systems and verification testing systems.

At present, the methods commonly used for preparing test standard reference substances are collectively referred to as fluorescent PCR amplification methods, mainly including: 1) mixing template DNA of different genotypes, PCR amplification, screening out samples containing different lengths of target fragments to be amplified, and carrying out Genomic DNA extraction, adjust the final concentration of each genomic DNA to be consistent, mix equal volumes, design fluorescently labeled PCR primers and amplify the mixed template, after fine-tuning the template concentration, the amplified product can be used as a standard; 2 ) Fluorescent PCR products of different lengths are mixed as a typing standard substance, that is, the preparation of a standard is carried out by mixing and adjusting the concentration of each PCR single template amplification product; 3) The secondary amplification method is to use the leveled amplification product as a standard substance DNA templates were re-amplified for standard preparation. Use the material containing the standard as a template, dilute 10 ³ to 10 ⁷ times, and re-amplify.

The main shortcoming of existing preparation method is as follows:

1. Pre-sample is easily restricted by the pre-batch sample collection, inspection, and screening process, including the heavy workload of sample collection, long time consumption, and high cost of typing inspection, resulting in long time-consuming and low efficiency in the overall preparation process.

2. The characteristics and performance of the prepared standard must depend on the sequence characteristics of the relevant samples, lack of flexibility, and it is difficult to meet the preparation requirements for the preparation of high-quality, high-standard, and high-flexibility test standards.

3. The operation is cumbersome and the leveling is difficult

4. The quality of standard control reagents is difficult to control

5. Strong dependence on samples, unable to achieve sustainable acquisition, greatly increasing the workload of the production process.

Contents of the invention

In view of the deficiencies in the prior art, the present invention aims to provide a method for preparing a special allele standard control reagent for genetic analyzer testing, using cloning technology to clone the column alleles of a specific STR site series into a selected cloning vector, and then Fluorescence amplification is performed on the above-mentioned carrier by multi-template compound amplification technology to realize sustainable preparation of allele standard control reagents.

In order to achieve the above object, the present invention adopts the following technical solutions:

A method for preparing a special allele standard control reagent for genetic analyzer testing, comprising the steps of:

S1 collects a large number of group samples, and obtains blood or saliva samples of a set number of people;

S2 performs STR typing detection on all samples collected in step S1, and determines the STR typing of each sample;

S3 selects samples containing corresponding typed alleles according to the STR typing results obtained in step S2;

Establishment of S4 allelic clone library: In the samples selected in step S3, the clones of all alleles are obtained by direct cloning for loci with complete alleles; The method of point-directed mutagenesis after cloning is used to establish the allelic clone library;

After the establishment of the S5 allele clone library, the preparation of fluorescent standards began: first, design and synthesize specific fluorescent primers for the corresponding sites, serially dilute the selected plasmid DNA, and perform fluorescent PCR amplification to explore the specificity of the plasmid DNA. After the optimal concentration, the templates are mixed and leveled, and after the concentration of each plasmid DNA is determined, the multi-template fluorescence composite PCR is amplified and detected, and after the inspection, it is purified and stored in aliquots.

It should be noted that in step S4, the clones of all alleles are obtained by direct cloning for the loci with complete alleles, and the specific process is as follows:

4.1.1) Designing primers to perform PCR amplification on the screened homozygotes, amplified products were separated by agarose gel electrophoresis, stained, and the product was recovered and purified by gel and inserted into a plasmid vector;

4.1.2) introducing the recombinant plasmid into Escherichia coli and culturing the Escherichia coli to obtain a large number of copies of a single DNA molecule;

4.1.3) Sequence and screen the positive clones with the correct sequence and perform DNA sequencing analysis on the recombinant plasmid with plasmid universal primers to confirm the sequence information of the inserted fragment, classify the inserted allelic fragments according to the number of repetitions of the repeat unit, and establish length allelic clone library.

It should be noted that in step S4, the method of cloning first and then site-directed mutagenesis is adopted for the allelic incomplete site to establish the allelic clone library, and the specific process is as follows:

4.2.1) Design cloning primers to carry out PCR amplification on the selected typing samples, and the amplified products are separated by agarose gel electrophoresis, stained, and the product gel is recovered and purified and then inserted into a plasmid vector;

4.2.2) introducing the recombinant plasmid into Escherichia coli and culturing the Escherichia coli to obtain a large number of copies of a single DNA molecule;

4.2.3) Sequence and screen the positive clones with correct sequences and perform DNA sequencing on the recombinant plasmids with plasmid universal primers, analyze and confirm the sequence information of the inserted fragments, classify the inserted allelic fragments according to the number of repetitions of the repeat unit, and then use the core Sequence-directed mutagenesis technology obtains a series of different allelic clones with a specified core sequence repetition number, and then establishes a library of all allele clones at this site.

It should be noted that, in step S5, the specific process of performing fluorescent PCR amplification to explore the optimal concentration of plasmid DNA is as follows:

The specific fluorescent primers of 0.2uM were used to amplify plasmid DNA of different concentrations, the amplified product was detected by a genetic analyzer, and the genemapper software was used for analysis. The optimum concentration of the plasmid DNA should meet the obtained fraction. The relative fluorescence intensity of the peaks in the pattern spectrum, that is, the RFU value, is kept within 2000-9000, and the peak shape is sharp, and there is no phenomenon that the template concentration cannot be judged according to the peak height (such as overflow peaks, flat peaks, and non-specificity caused by random amplification). Fluorescent peaks, etc.) appear.

The beneficial effects of the present invention are: the outstanding stability and mass production of the high-quality typing reference substance prepared based on the method are incomparable with traditional methods, and mainly have the following advantages:

1. The target gene can be permanently preserved through the recombinant plasmid, and the establishment of the recombinant plasmid library can realize the permanent identity of the typing standard;

2. A large number of allele fragments can be obtained by PCR amplification using a small amount of recombinant plasmid loaded with a specific gene fragment as a template. Because the DNA of the recombinant plasmid is circular, the alleles loaded on the plasmid show extremely high amplification efficiency during PCR amplification;

3. Make the production process of DNA standards more controllable and reduce the difference between batches of standards;

4. After the target gene is loaded on the plasmid, the common primers for plasmid sequencing can be used to complete the analysis of its sequence structure.

Description of drawings

Fig. 1 is the overall schematic flow chart of the present invention;

Fig. 2 is the schematic flow chart of embodiment 1 of the present invention;

Fig. 3a and Fig. 3b are the detection result diagrams of the D13S317-FAM allele standard control reagent prepared in Example 1 of the present invention on the GA118-16A genetic analyzer;

Figure 4 is a preliminary analysis result diagram of GeneMapper ID 3.2 at the D8S1179 site in Example 2 of the present invention;

Figure 5a and Figure 5b are the analysis results of GeneMapper ID3.2 at the D8S1179 site in Example 2 of the present invention (after the compilation of the Panel and bin files).

Detailed ways

The present invention will be further described below in conjunction with the accompanying drawings. It should be noted that the following examples are based on the premise of this technical solution, and provide detailed implementation and specific operation process, but the protection scope of the present invention is not limited to the present invention. Example.

As shown in Figure 1, a kind of genetic analyzer tests special-purpose allele standard control reagent preparation method, comprises the steps:

S1 collects a large number of group samples, and obtains blood or saliva samples of a set number of people;

S2 performs STR typing detection on all samples collected in step S1, and determines the STR typing of each sample;

S3 selects samples containing corresponding alleles according to the STR typing results obtained in step S2; taking the human locus D8S1179 as an example, this locus contains 12 types "7", "8", and "9", "10", "11", "12", "13", "14", "15", "16", "17", "18", then these genotypes are distributed in different human in the sample. After the typing of each individual is determined through step S2, it is determined which samples are contained in the above-mentioned 12 types of alleles, and then those samples need to be selected for subsequent gene cloning.

Establishment of S4 allelic clone library: In the samples selected in step S3, the clones of all alleles are obtained by direct cloning for loci with complete alleles; The method of point-directed mutagenesis after cloning is used to establish the allelic clone library;

After the establishment of the S5 allele clone library, the preparation of fluorescent standards began: first, design and synthesize specific fluorescent primers for the corresponding sites, serially dilute the selected plasmid DNA, and perform fluorescent PCR amplification to explore the specificity of the plasmid DNA. After the optimal concentration, the templates are mixed and leveled, and after the concentration of each plasmid DNA is determined, the multi-template fluorescence composite PCR is amplified and detected, and after the inspection, it is purified and stored in aliquots.

It should be noted that in step S4, the clones of all alleles are obtained by direct cloning for the loci with complete alleles, and the specific process is as follows:

4.1.1) Designing primers to perform PCR amplification on the screened homozygotes, amplified products were separated by agarose gel electrophoresis, stained, and the product was recovered and purified by gel and inserted into a plasmid vector;

4.1.2) introducing the recombinant plasmid into Escherichia coli and culturing the Escherichia coli to obtain a large number of copies of a single DNA molecule;

4.1.3) Sequence and screen the positive clones with the correct sequence and perform DNA sequencing analysis on the recombinant plasmid with plasmid universal primers to confirm the sequence information of the inserted fragment, classify the inserted allelic fragments according to the number of repetitions of the repeat unit, and establish length allelic clone library.

It should be noted that in step S4, the method of cloning first and then site-directed mutagenesis is adopted for the allelic incomplete site to establish the allelic clone library, and the specific process is as follows:

4.2.1) Design cloning primers to carry out PCR amplification on the selected typing samples, and the amplified products are separated by agarose gel electrophoresis, stained, and the product gel is recovered and purified and then inserted into a plasmid vector;

4.2.2) introducing the recombinant plasmid into Escherichia coli and culturing the Escherichia coli to obtain a large number of copies of a single DNA molecule;

4.2.3) Sequence and screen the positive clones with correct sequences and perform DNA sequencing on the recombinant plasmids with plasmid universal primers, analyze and confirm the sequence information of the inserted fragments, classify the inserted allelic fragments according to the number of repetitions of the repeat unit, and then use the core Sequence-directed mutagenesis technology obtains a series of different allelic clones with a specified core sequence repetition number, and then establishes a library of all allele clones at this site.

It should be noted that, in step S5, the specific process of performing fluorescent PCR amplification to explore the optimal concentration of plasmid DNA is as follows:

The specific fluorescent primers of 0.2uM were used to amplify plasmid DNA of different concentrations, the amplified product was detected by a genetic analyzer, and the genemapper software was used for analysis. The optimum concentration of the plasmid DNA should meet the obtained fraction. The relative fluorescence intensity of the peaks in the pattern spectrum, that is, the RFU value, is kept within 2000-9000, and the peak shape is sharp, and there is no phenomenon that the template concentration cannot be judged according to the peak height (such as overflow peaks, flat peaks, and non-specificity caused by random amplification). Fluorescent peaks, etc.) appear.

Example 1

Preparation of STR site-D13S317-FAM fluorescently labeled (blue) standard (complete alleles)

As shown in Figure 2, the preparation method of the present embodiment comprises the following steps:

(1) Design primer 1 (cloning primer) using the D13S317 locus of the human genome as a template. The primer sequence is as follows:

Forward primer: F-5'ATATTGTGATAATTTAAGGAGGCA-3',

Reverse primer: R-5'TTTTAAGAAAGTAAACAGGCATCA-3'

(2) Use the above primers to amplify DNA from human blood samples containing 7, 8, 9, 10, 11, 12, and 13 repeat units, and the amplification conditions are as follows:

95°C, 11min;

94℃, 30s; 59℃, 2min; 72℃, 1min; 28 cycles;

60℃, 60min;

4 ℃, permanent storage.

(3) The amplified product was subjected to agarose gel electrophoresis and the gel recovery kit was used for gel recovery

3.1) The amplified product was evenly mixed with 10×loading buffer, and DNA was separated using 1% agarose gel (EB staining);

3.2) UV imaging and cutting the gel containing the target fragment into a 1.5ml centrifuge tube;

3.3) Use the agarose DNA recovery kit (TianGen company) to perform the gel recovery operation according to the operation manual;

(4) The amplified fragment is connected to the cloning vector

4.1) Select the T vector plasmid vector PMD-18T (TAKARA company);

4.2) Prepare connection system;

4.3) Connect at 25°C for 60 minutes;

(5) Transform Escherichia coli competent

5.1) Take the competent cells and place them in an ice bath. After the competent cells are melted, add the above ligation product to 100ul DH5α E. coli competent cell suspension, gently blow and beat to mix, and ice-bath for 30min.

5.2) Heat shock at 42°C for 45 seconds, quickly transfer the centrifuge tube to ice, and let it stand for 2-3 minutes;

5.3) Add 450ul of sterile ampicillin-free LB medium to the centrifuge tube, mix well, place at 37°C, shake and culture for 45min to recover the bacteria;

5.4) Take 100ul of transformed competent cells, spread evenly on LB solid medium containing ampicillin, and culture at 37°C for 12-16 hours

(6) Pick positive clones and shake overnight. ;

(7) Plasmid extraction (TianGen plasmid extraction kit, just follow the operating instructions of the corresponding kit)

(8) Plasmid sequencing

(9) Keep the recombinant plasmid with the correct sequence

(10) Quantification of plasmid DNA

(11) Preparation of allele standard controls

11.1) Dilute a single recombinant plasmid containing the correct allele insert to 1ng/μl with TE buffer

11.2) Design and synthesize FAM-labeled primers (Shanghai Sangong).

The primer sequences are as follows:

6-FAM 5'-TCTTTAGTGGGCATCCGTGAC

5'-AACTTGGGTTGAGCCATAGG

11.3) Dilute the purified product 10, 102, 103, 104, 105, and 106 times respectively, and use the diluted product as a template for PCR amplification

20μl amplification system:

10×Reaction Buffer 2.0μl

MgCl2 (25mmol/L) 1.2μl

10μmol/L upstream and downstream primers 0.6μl each

dNTP (10mmol/L)0.4μl

Taq polymerase (5U/μl) 0.2μl

Template 2μl

ddH2O 13μl

PCR reaction conditions:

95℃, 11min

94℃, 30s; 59℃, 2min; 72℃, 1min; 28 cycles

60℃, 60min

4 ℃, permanent storage.

(12) Perform detection on the GA118-16A instrument, obtain preliminary detection results, and adjust the template concentration according to the detection results

The final composite template adjustment method is shown in Table 1:

Table 1

Then add 1ml TE, shake and mix well, take 2ul, and make a 20ul amplification system.

(13) Amplify according to the above conditions, perform product purification, and detect GA118-16A according to the operating instructions. Store at -20°C for device testing.

The detection results of the STR site-D13S317-FAM fluorescently labeled (blue) standard substance prepared in this example on the GA118-16A genetic analyzer are shown in Figure 3a and Figure 3b. The test results show that the allele standard control reagent has a complete number of allele peaks, correct fragment length difference (4bp difference), uniform peak height distribution, sharp peak shape, no penetration peak, shadow band, non-specific peak, and moderate peak height , showing superior performance.

Example 2

The preparation of the allele typing standard substance of D8S1179 locus comprises the following steps:

(1) Design cloning primers using the D8S1179 locus as a template (sample alleles are incomplete):

D8S1179-Non-F: 5'-AGCCACCGCACCCCAGCCTGTT-3'

D8S1179-Non-R: 5'-CCATGTTAGCCAGGATGGTC-3'

(2) Using the above primers to amplify the human blood sample DNA, the amplification conditions are as follows:

95℃, 11min

94℃, 30s; 59℃, 2min; 72℃, 1min; 28 cycles

60℃, 60min

4 ℃, permanent storage.

(3) The amplified product was subjected to agarose gel electrophoresis and the gel recovery kit was used for gel recovery

3.1) Mix the amplified product with 10×loading buffer evenly, and use 1% agarose gel (EB staining) for DNA separation

3.2) UV imaging and cut the gel containing the target fragment into a 1.5ml centrifuge tube

3.3) Use the agarose DNA recovery kit (TianGen company) to perform the gel recovery operation according to the operation manual

(4) The amplified fragment is connected to the cloning vector

4.1) Select the T vector plasmid vector PMD-18T (TAKARA company)

4.2) Prepare connection system

Connect at 25°C for 60min

(5) Transform Escherichia coli competent

5.1) Take the competent cells and place them in an ice bath. After the competent cells are melted, add the above ligation product to 100ul DH5α E. coli competent cell suspension, gently blow and beat to mix, and ice-bath for 30min.

5.2) Heat shock at 42°C for 45 seconds, quickly transfer the centrifuge tube to ice, and let it stand for 2-3 minutes

5.3) Add 450ul sterile ampicillin-free LB medium into the centrifuge tube, mix well, place at 37°C, shake and culture for 45min to recover the bacteria

5.4) Take 100ul of transformed competent cells, spread evenly on LB solid medium containing ampicillin, and culture at 37°C for 12-16 hours

(6) Pick positive clones and shake overnight.

(7) Plasmid extraction (TianGen plasmid extraction kit, just follow the operating instructions of the corresponding kit)

(8) Plasmid sequencing

(9) Keep the recombinant plasmid with the correct sequence

(10) Based on this plasmid, perform site-directed mutation on its core STR repeat sequence, and construct a series of plasmids (D8S1179-7, D8S1179-8, D8S1179-9, D8S1179-10, D8S1179-11, D8S1179- 12, D8S1179-13, D8S1179-14, D8S1179-15, D8S1179-16, D8S1179-17, D8S1179-18)

(11) Quantification of plasmid DNA and preparation of allele standard controls

11.1) Dilute a single recombinant plasmid containing the correct allele insert to 1 ng/μl with TE buffer.

11.2) Design and synthesize FAM-labeled primers (Shanghai Sangong).

The primer sequences are as follows:

D8S1179-HEX:CCATTGCGTGAATATGCCTTA

D8S1179-REV:ATGTATTTTTGTATTTCATGTGTACATT

11.3) Dilute the purified product 10, 102, 103, 104, 105, and 106 times respectively, and use the diluted product as a template for PCR amplification

20μl amplification system:

10×Reaction Buffer 2.0μl

MgCl2 (25mmol/L) 1.2μl

10μmol/L upstream and downstream primers 0.6μl each

dNTP (10mmol/L)0.4μl

Taq polymerase (5U/μl) 0.2μl

Template 2μl

ddH2O 13μl

PCR reaction conditions:

95℃, 11min

94℃, 30s; 59℃, 2min; 72℃, 1min; 28 cycles

60℃, 60min

4 ℃, permanent storage.

(12) Perform detection on the GA118-16A instrument, obtain preliminary detection results, and adjust the template concentration according to the detection results.

The final composite template adjustment method is shown in Table 2:

Table 2

Then add 1ml TE, shake and mix well, take 2ul, and make a 20ul amplification system.

The amplified product was detected by a domestic genetic analyzer GA118-16A, and all detection operations were carried out according to the instructions of the equipment. After the detection of GA118-16A, the template concentration and amplification parameters were fine-tuned according to the preliminary results to obtain the allelic typing standard of the D8S1179 locus. The detection results of GA118-16A were analyzed by GeneMapper ID 3.2, as shown in Figure 4. The test results show that the allele peaks of the allele standard control reagent are complete, the peak height (RFU) difference of each allele can be controlled within 5%, the balance is extremely high, the peak shape is sharp, there is no double shoulder peak, No permeation peaks, shadow bands, non-specific peaks, and moderate peak heights, showing superior performance.

According to the length information of each allele of D8S1179, the Panel and bin files were compiled, and the gene Mapper was imported to obtain the typing results marked with the information of each allele, as shown in Figure 5a and Figure 5b. The test results show that the allele standard control reagent has complete allele peaks and the typing result is correct, which can fully meet the requirements of the allele standard control.

For those skilled in the art, various corresponding changes and modifications can be made according to the above technical solutions and concepts, and all these changes and modifications should be included in the protection scope of the claims of the present invention.

Claims (4)

1. A method for preparing a special-purpose allele standard control reagent for genetic analyzer testing, is characterized in that, comprises the steps: S1 collects a large number of group samples, and obtains blood or saliva samples of a set number of people; S2 performs STR typing detection on all samples collected in step S1, and determines the STR typing of each sample; S3 selects samples containing corresponding typed alleles according to the STR typing results obtained in step S2; Establishment of S4 allelic clone library: In the samples selected in step S3, the clones of all alleles are obtained by direct cloning for loci with complete alleles; The method of point-directed mutagenesis after cloning is used to establish the allelic clone library; After the establishment of the S5 allele clone library, the preparation of fluorescent standards began: first, design and synthesize specific fluorescent primers for the corresponding sites, serially dilute the selected plasmid DNA, and perform fluorescent PCR amplification to explore the specificity of the plasmid DNA. After the optimal concentration, the templates are mixed and leveled, and after the concentration of each plasmid DNA is determined, the multi-template fluorescence composite PCR is amplified and detected, and after the inspection, it is purified and stored in aliquots. 2. the method for preparing the special allele standard control reagent for genetic analyzer test according to claim 1, is characterized in that, in step S4, described for the complete locus of allele adopts the mode of direct cloning to obtain all alleles Gene cloning, the specific process is as follows: 4.1.1) Designing primers to perform PCR amplification on the screened homozygotes, amplified products were separated by agarose gel electrophoresis, stained, and the product was recovered and purified by gel and inserted into a plasmid vector; 4.1.2) introducing the recombinant plasmid into Escherichia coli and culturing the Escherichia coli to obtain a large number of copies of a single DNA molecule; 4.1.3) Sequence and screen the positive clones with the correct sequence and perform DNA sequencing analysis on the recombinant plasmid with plasmid universal primers to confirm the sequence information of the inserted fragment, classify the inserted allelic fragments according to the number of repetitions of the repeat unit, and establish length allelic clone library. 3. the preparation method of special allele standard control reagent for genetic analyzer test according to claim 1, is characterized in that, in step S4, described for the site of allelic insufficiency, adopts the method for cloning first and then site-directed mutation to carry out The establishment of the allelic clone library, the specific process is as follows: 4.2.1) Design cloning primers to carry out PCR amplification on the selected typing samples, and the amplified products are separated by agarose gel electrophoresis, stained, and the product gel is recovered and purified and then inserted into a plasmid vector; 4.2.2) introducing the recombinant plasmid into Escherichia coli and culturing the Escherichia coli to obtain a large number of copies of a single DNA molecule; 4.2.3) Sequence and screen the positive clones with correct sequences and perform DNA sequencing on the recombinant plasmids with plasmid universal primers, analyze and confirm the sequence information of the inserted fragments, classify the inserted allelic fragments according to the number of repetitions of the repeat unit, and then use the core Sequence-directed mutagenesis technology obtains a series of different allelic clones with a specified core sequence repetition number, and then establishes a library of all allele clones at this site. 4. genetic analyzer test according to claim 1 special-purpose allelic standard control reagent preparation method, it is characterized in that, in step S5, described carrying out fluorescent PCR amplification is to grope the concrete flow process of plasmid DNA optimal concentration: The specific fluorescent primers of 0.2uM were used to amplify plasmid DNA of different concentrations, the amplified product was detected by a genetic analyzer, and the genemapper software was used for analysis. The optimum concentration of the plasmid DNA should meet the obtained fraction. The relative fluorescence intensity of the peaks in the pattern spectrum, that is, the RFU value, remained within 2000-9000, and the peak shape was sharp, and there was no phenomenon that the template concentration could not be judged based on the peak height.