阅读说明：本技术 一种八色荧光光谱校准试剂及其制备方法 (Eight-color fluorescence spectrum calibration reagent and preparation method thereof ) 是由 姜伯玮 张黎明 王峰 荣海博 赵怡鹤 张涛 金川 陈力 于 2021-09-26 设计创作，主要内容包括：本发明公开了一种八色荧光光谱校准试剂及其制备方法,采用人工设计合成的8个模板DNA,每个模板DNA模板分别包含1对引物的结合位点,并根据每个片段的预设长度,可实现片段长度与荧光精确控制。每对引物中的上游引物的5’端分别采用8种不同荧光标记,在一个特定反应体系内进行复合扩增,可同时获得含有八种荧光产物的荧光光谱校准试剂,无需进行单色荧光片段的混合和调配即可直接应用于荧光光谱校准。该方法极大的简化了荧光标准物的制备工艺流程,避免了复杂繁琐的操作。本发明的制备方法可以稳定的获得该标准物质,具有效率高,操作简便的特点。(The invention discloses an eight-color fluorescence spectrum calibration reagent and a preparation method thereof, 8 template DNAs which are artificially designed and synthesized are adopted, each template DNA template respectively comprises binding sites of 1 pair of primers, and the accurate control of the length of a fragment and fluorescence can be realized according to the preset length of each fragment. The 5' end of the upstream primer in each pair of primers respectively adopts 8 different fluorescent labels, composite amplification is carried out in a specific reaction system, the fluorescence spectrum calibration reagent containing eight fluorescent products can be obtained simultaneously, and the fluorescence spectrum calibration reagent can be directly applied to fluorescence spectrum calibration without mixing and blending monochromatic fluorescent fragments. The method greatly simplifies the preparation process flow of the fluorescent standard substance and avoids complex and fussy operation. The preparation method can stably obtain the standard substance and has the characteristics of high efficiency and simple and convenient operation.)

1. A preparation method of an eight-color fluorescence spectrum calibration reagent is characterized by comprising the following specific steps:

s1, artificially synthesizing 8 template DNAs, wherein each template DNA comprises binding sites of different primers; the sequence information of 8 template DNAs are shown in SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8 is shown in the specification;

s2, designing and determining 8 pairs of fluorescent primers, wherein sequence information of the fluorescent primer pair 1, the fluorescent primer pair 2, the fluorescent primer pair 3, the fluorescent primer pair 4, the fluorescent primer pair 5, the fluorescent primer pair 6, the fluorescent primer pair 7 and the fluorescent primer pair 8 is respectively SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20. SEQ ID NO: 21 and SEQ ID NO: 22. SEQ ID NO: 23 and SEQ ID NO: 24, and the 5' end fluorescent labels are FAM, HEX, NED, NH598, NH618, NH650, NH635 and NH670 respectively;

s3, mixing 8 artificially synthesized template DNAs obtained in the step S1 to obtain a template mixture;

s4, mixing the 8 pairs of fluorescent primer pairs obtained in the step S2 to obtain a primer mixture;

and S5, mixing the PCR buffer solution, the template mixture obtained in the step S3 and the primer mixture obtained in the step S4, and performing PCR amplification to obtain the eight-color fluorescence spectrum calibration reagent.

2. The method according to claim 1, wherein in step S3, the template mixture contains 10. mu.L of each of 8 pieces of template DNA and 20. mu.L of PCR-grade water in a 100. mu.L system.

3. The method according to claim 2, wherein the concentrations of the 8 pieces of template DNA participating in mixing are all 1 ng/. mu.L.

4. The method according to claim 1, wherein in step S4, the primer mixture comprises 6.5. mu.L of fluorescent primer pair 1, 6.25. mu.L of fluorescent primer pair 2, 6. mu.L of fluorescent primer pair 3, 5. mu.L of fluorescent primer pair 4, 5. mu.L of fluorescent primer pair 5, 4.0. mu.L of fluorescent primer pair 6, 4.5. mu.L of fluorescent primer pair 7, 3.25. mu.L of fluorescent primer pair 8, and 59.5. mu.L of PCR-grade water or TE^-1。

5. The method according to claim 4, wherein the concentration of each of the 8 pairs of fluorescent primer pairs involved in the mixing is 2. mu.M.

6. The method according to claim 1, wherein the PCR amplification system comprises 4. mu.L of 5 XPCR reaction buffer, 10. mu.L of primer mixture, 1. mu.L of template mixture and 5. mu.L of PCR-grade water in a 20. mu.L system in step S5.

7. The method of claim 1, wherein in step S5, PCR cycling parameters are first activated by initial incubation at 95 ℃ for 5 minutes, then cycled by reaction at 95 ℃ for 20S, reaction at 59 ℃ for 1min, reaction at 72 ℃ for 30S for 27-28 times, final extension at 60 ℃ for 20 minutes, and finally incubated at 4 ℃.

8. The eight-color fluorescence spectrum calibration reagent prepared by the preparation method of any one of claims 1 to 7.

Technical Field

The invention relates to the technical field of DNA detection, in particular to an eight-color fluorescence spectrum calibration reagent and a preparation method thereof.

Background

The DNA detection technology becomes one of the most important technical means in the field of modern court science, and is the most effective individual recognition tool in the major events of treating various cases, serious accidents, natural disasters and the like. DNA inspection equipment and DNA detection reagents are two key elements for developing DNA detection work.

The most common detection devices in the domestic DNA detection laboratory are mainly 3130, 3130xl, 3500 and 3500xl type genetic analyzers. Instruments and equipment in the field of DNA detection in international court science have undergone a development history of four, five to six colors.

The earliest DNA reagent system for DNA detection was a four-site multiplex amplification system of TH01, FES \ FPS, vWA and F13a1 established by the british court scientific service (FSS), which was called the first generation multiplex amplification system, and the matching probability was nearly one in ten thousand. In 1994, FSS developed a second generation multiplex amplification System (SGM) comprising six polymorphic STR loci and a sex gene, TH01, vWA, FGA, D8S1179, D18S51, D21S11 and Amelogenin (enamel gene), respectively, with a match probability of approximately one-half of a million. In 1998, four new STR loci were added: D19S433, D2S1338, D16S539 and D3S1358, the matching probability can reach one billion. In order to meet the needs of handling, FBI in the united states has established a Combined DNA Index System (CODIS) consisting of 13 STR loci, TPOX, D3S1358, TH01, vWA, FGA, D7S820, D21S11, D18S51, D5S818, D13S317, CSF1PO, D16S539 and D8S1179, and the average random matching probability of unrelated individuals is greater than one billion. In 2001, AmpFlSTR identifier kit was introduced by ABI corporation, usa, for the first time using a five-color fluorescent detection system in which four colors mark the PCR product and the remaining one fluorescent color is used to detect an internal standard to correct the electrophoretic migration of the PCR product fragments. After 2013, 24-locus six-color fluorescent DNA test kit GlobalFiler, including 25-locus kit VeriFiler and VeriFiler Plus, was successively introduced by ABI company in the United states. At the same time, the Promega corporation in the United states also introduced the PowerPlex Fusion six-color fluorescent kit containing 24 sites, all based on an imported genetic Analyzer. Several related units and reagent companies in China also put forward various STR multiplex amplification detection kits with five colors and six colors, such as Typer21(21 site), Typer Y36(36 site) and the like of the national department of public Security evidence identification center, EX25(25 site), Y37(37 site) kit of Med, Pan Global (27 site) of Heisha, PathFinder Plus (37Y sites) kit and the like. Therefore, the development of DNA detection technology has the characteristic that the detected fluorescence number is gradually increased.

In recent years, the development of DNA inspection technology in the national court science is rapid, multiple breakthroughs are made in the aspects of localization of key instruments and equipment and reagents for DNA inspection, and the first international genetic analyzer, composite amplification reagent and analysis software capable of realizing eight-color fluorescence inspection are innovatively developed on the basis of five-color and six-color fluorescence technologies in China, so that the technical lead in the field is realized. The eight-color fluorescent spectrum calibration reagent is a necessary matching reagent for applying the eight-color fluorescent STR multiplex amplification test kit to a domestic genetic analyzer, and is an important link that the domestic eight-color fluorescent reagent can be tightly combined with a domestic forensic DNA special detection platform. A plurality of different DNA detection kits released by a plurality of companies at home and abroad are all provided with special fluorescent spectrum calibrators, and the preparation technical method is secret. Therefore, development of research and development work of the autonomous eight-color fluorescence spectrum calibration reagent is urgently required.

Chinese patent application ZL201110201443.3 discloses a five-color fluorescent matrix standard substance and a preparation method and a special primer composition thereof, wherein 9947A standard DNA is used as a template, a plurality of pairs of primers are adopted for composite amplification, but shadow bands (Stutter) are easily generated in the amplification process, so that a plurality of mixed peaks are contained in a fluorescent spectrum calibration reagent, the quality of the spectrum calibration reagent is greatly influenced, and the quality number is reduced. In addition, most of the traditional fluorescent spectrum calibration reagents are prepared by adopting single-fluorescence fragment amplification, purification, mixing and blending methods, and the preparation process is complex and tedious.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims to provide an eight-color fluorescence spectrum calibration reagent and a preparation method thereof.

In order to achieve the purpose, the invention adopts the following technical scheme:

a preparation method of an eight-color fluorescence spectrum calibration reagent comprises the following specific processes:

s1, artificially synthesizing 8 template DNAs, wherein each template DNA comprises binding sites of different primers; the sequence information of 8 template DNAs are shown in SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: 8 is shown in the specification;

s2, designing and determining 8 pairs of fluorescent primers, wherein sequence information of the fluorescent primer pair 1, the fluorescent primer pair 2, the fluorescent primer pair 3, the fluorescent primer pair 4, the fluorescent primer pair 5, the fluorescent primer pair 6, the fluorescent primer pair 7 and the fluorescent primer pair 8 is respectively SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20. SEQ ID NO: 21 and SEQ ID NO: 22. SEQ ID NO: 23 and SEQ ID NO: 24, and the 5' end fluorescent labels are FAM, HEX, NED, NH598, NH618, NH650, NH635 and NH670 respectively;

s3, mixing 8 artificially synthesized template DNAs obtained in the step S1 to obtain a template mixture;

s4, mixing the 8 pairs of fluorescent primer pairs obtained in the step S2 to obtain a primer mixture;

and S5, mixing the PCR buffer solution, the template mixture obtained in the step S3 and the primer mixture obtained in the step S4, and performing PCR amplification to obtain the eight-color fluorescence spectrum calibration reagent.

Further, in step S3, the template mixture contains 8 pieces of template DNA each 10. mu.L and PCR-grade water 20. mu.L in a 100. mu.L system.

Further, the concentrations of the 8 pieces of template DNA participating in the mixing were all 1 ng/. mu.L.

Further, in step S4, the primer mixture contains 6.5. mu.L of fluorescent primer pair 1, 6.25. mu.L of fluorescent primer pair 2, 6. mu.L of fluorescent primer pair 3, 5. mu.L of fluorescent primer pair 4, 5. mu.L of fluorescent primer pair 5, 4.0. mu.L of fluorescent primer pair 6, 4.5. mu.L of fluorescent primer pair 7, 3.25. mu.L of fluorescent primer pair 8, and 59.5. mu.L of PCR-grade water or TE^-1。

Further, the concentration of each of the 8 pairs of fluorescent primer pairs involved in the mixing was 2. mu.M.

Further, in step S5, the PCR amplification system comprises 4. mu.L of 5 XPCR reaction buffer, 10. mu.L of primer mixture, 1. mu.L of template mixture and 5. mu.L of PCR-grade water, based on a 20. mu.L system.

Further, in step S5, PCR cycling parameters were first activated by initial incubation at 95 ℃ for 5 minutes, followed by cycling through 95 ℃ for 20S, 59 ℃ for 1min, and 72 ℃ for 30S, for 27-28 times, and then final extension at 60 ℃ for 20 minutes, and finally incubation at 4 ℃.

The invention also provides the eight-color fluorescence spectrum calibration reagent prepared by the preparation method.

The invention has the beneficial effects that: the invention adopts 8 template DNAs which are artificially designed and synthesized, each template DNA respectively comprises 1 pair of primer binding sites, and the accurate control of the fragment length and the fluorescence can be realized according to the preset length of each fragment. The 5' end of the upstream primer in each pair of primers respectively adopts 8 different fluorescent labels, composite amplification is carried out in a specific reaction system, the fluorescence spectrum calibration reagent containing eight fluorescent products can be obtained simultaneously, and the fluorescence spectrum calibration reagent can be directly applied to fluorescence spectrum calibration without mixing and blending monochromatic fluorescent fragments. The method greatly simplifies the preparation process flow of the fluorescent standard substance and avoids complex and fussy operation. The preparation method can stably obtain the standard substance and has the characteristics of high efficiency and simple and convenient operation.

Drawings

FIG. 1 is a diagram showing the results of detection of Template1 and FAM-labeled primers in example 1 of the present invention;

FIG. 2 is a diagram showing the detection results of Template2 and HEX-labeled primers in example 1 of the present invention;

FIG. 3 is a diagram showing the detection results of Template3 and NED-labeled primers in example 1 of the present invention;

FIG. 4 is a diagram showing the detection results of Template4 and NH 598-labeled primers in example 1 of the present invention;

FIG. 5 is a diagram showing the detection results of Template5 and NH618 labeled primers in example 1 of the present invention;

FIG. 6 is a diagram showing the detection results of Template6 and NH635 labeled primers in example 1 of the present invention;

FIG. 7 is a diagram showing the detection results of Template7 and NH650 labeled primers in example 1 of the present invention;

FIG. 8 is a diagram showing the detection results of Template8 and NH670 labeled primer in example 1 of the present invention;

FIG. 9 is a diagram showing the results of detection in example 2 of the present invention;

FIG. 10 is a schematic diagram showing the passage of the eight-color fluorescence spectrum reagent for spectrum calibration in example 3 of the present invention;

FIG. 11 is a schematic view of the results (fragments) of eight-color spectrum correction of a GA118-24B type genetic analyzer in example 3 of the present invention;

FIG. 12 is a graph showing the results of eight-color spectrum correction (spectral distribution) by a GA118-24B type genetic analyzer in example 3 of the present invention;

FIG. 13 is a spectrum of eight-color fluorescence detection after calibration of eight-color fluorescence in example 4 of the present invention.

Detailed Description

The present invention will be further described with reference to the accompanying drawings, and it should be noted that the present embodiment is based on the technical solution, and the detailed implementation and the specific operation process are provided, but the protection scope of the present invention is not limited to the present embodiment.

Example 1

The embodiment provides a preparation method of an eight-color fluorescence spectrum calibration reagent, which comprises the following specific processes:

s1, artificially synthesizing 8 template DNAs, wherein each template DNA comprises binding sites of different primers, and the 8 template DNAs are connected with a PMD-18T carrier through T/A; the sequence information of 8 template DNAs are shown in SEQ ID NO: 1. SEQ ID NO: 2. SEQ ID NO: 3. SEQ ID NO: 4. SEQ ID NO: 5. SEQ ID NO: 6. SEQ ID NO: 7. SEQ ID NO: shown in fig. 8.

S2, designing and determining 8 pairs of fluorescent primers, wherein the sequence information of the 8 pairs of fluorescent primers is respectively SEQ ID NO: 9 and SEQ ID NO: 10, SEQ ID NO: 11 and SEQ ID NO: 12, SEQ ID NO: 13 and SEQ ID NO: 14, SEQ ID NO: 15 and SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, SEQ ID NO: 19 and SEQ ID NO: 20. SEQ ID NO: 21 and SEQ ID NO: 22. SEQ ID NO: 23 and SEQ ID NO: 24, as shown in table 1.

TABLE 1

Fluorescence labeling and synthesis were performed according to the primer sequences, purification was performed by HPLC, and the single primers were diluted to 100 μ M, and then the primer pair was diluted to 2 μ M working concentration, and the PCR system is shown in table 2 below.

TABLE 2

The amplification procedure is shown in table 3:

TABLE 3

Mixing 1 μ L of amplification product with 10 μ L of Hi-Di Formamide, performing capillary electrophoresis detection with GA118-24B genetic analyzer, and analyzing with GA-Marker software to obtain detection map, wherein the amplification and detection results of each primer are shown in FIGS. 1-8.

S3, mixing 8 template DNAs artificially synthesized in the step S1 according to the concentration ratio shown in the table 4 to prepare a template mixture;

TABLE 4

Composition of	Volume (100. mu.L system)
		SEQ ID NO：1(1ng/μL)	10μL
SEQ ID NO：2(1ng/μL)	10μL
		SEQ ID NO：3(1ng/μL)	10μL
SEQ ID NO：4(1ng/μL)	10μL
		SEQ ID NO：5(1ng/μL)	10μL
SEQ ID NO：6(1ng/μL)	10μL
		SEQ ID NO：7(1ng/μL)	10μL
SEQ ID NO：8(1ng/μL)	10μL
		PCR grade water	20μL

S4, 8 pairs of fluorescent primer pairs obtained in step S2 were prepared according to the preparation method shown in Table 5 to obtain a primer mixture.

TABLE 5

Composition of	Volume (100. mu.L system)	Final concentration in PCR System
			Primer set 1 (2. mu.M)	6.5μL	0.065μM
Primer set 2 (2. mu.M)	6.25μL	0.0625μM
			Primer pair 3 (2. mu.M)	6μL	0.06μM
Primer set 4 (2. mu.M)	5μL	0.05μM
			Primer set 5 (2. mu.M)	5μL	0.05μM
Primer pair 6 (2. mu.M)	4.0μL	0.04μM
			Primer pair 7 (2. mu.M)	4.5μL	0.045μM
Primer pair 8 (2. mu.M)	3.25μL	0.0325μM
			PCR grade water or TE-1	Make up to 100 mu L

S5, mixing the PCR buffer solution, the template mixture obtained in the step S3 and the primer mixture obtained in the step S2, and amplifying under the conditions of the PCR cycle parameters shown in Table 7; the PCR amplification system is shown in Table 6.

TABLE 6

TABLE 7

Mixing 1 μ L of the amplification product obtained in step S6 with 10 μ L of formamide, denaturing at 95 ℃ for 3 minutes, performing capillary electrophoresis detection by using a GA118-24B genetic analyzer (first institute of public Security), and analyzing by GA-Marker software (first institute of public Security) to obtain a detection map, wherein the detection result is shown in FIG. 9.

The result shows that the method of the embodiment can simultaneously obtain a high-quality fluorescence calibration mixture through one-step amplification, 8 different fluorescence peaks are formed, the peak type is sharp, the fluorescence fragment is complete, the peak height completely meets the requirement of a fluorescence calibration reagent, and no non-specific impurity peak is generated.

Example 2

The present embodiment provides a method for generating an eight-color fluorescence calibration file.

The eight-color fluorescent calibration reagent prepared in example 1 needs to be detected by a domestic genetic analyzer GA118-24B to generate a corresponding spectral calibration file for detecting the corresponding eight-color fluorescent labeled fluorescent PCR product.

Taking 25 mu L of eight-color fluorescent calibration reagent, mixing with 225 mu L of formamide, fully and uniformly mixing, denaturing at 95 ℃ for 5min and at-20 ℃ for 5min, subpackaging 10 mu L of each 24 holes in three rows (A1-H3) of a 96-hole plate, and detecting by using a GA118-24B type genetic analyzer according to an operation instruction, wherein the specific parameters are as follows:

selection of T8 fluorescent channel mode

The length of the capillary is 36 cm;

electrophoresis conditions: the temperature is 60 ℃, the glue pouring time is 15s, and the electrophoresis voltage is 15000V;

sample introduction voltage: 1.2 kv;

sample introduction time: 10 s;

electrophoresis time 1500s

Starting 'Genetic Analyzer Data Collection Software V1.0', selecting 'new form', popping up 'first step of new form' window, and naming '8 dye-matrix' in 'sample form name'; "application type" selection "SpectrallCal"; click on "next". In the "second step of the new form" window, click on the "A1 sample name" cell, hold the mouse and drag down to the "H3 sample name" cell, at which point all cells between the "A1 sample name" and the "H3 sample name" have been selected. Clicking the right mouse button to select the selected cells of "auto fill [ serial number ] (a)", "sample name" and "priority setting" will automatically fill. Clicking on the "A1 dye set" cell selects "T8" and the same column of cells will automatically fill. Clicking on the "A1 run model" cell selects "SpectraCal _ POP7_ Default" (selection according to the gel used), and the same column of selected cells will automatically fill. Clicking on the "A1 spectral calibration standard" cell selects "MtxStd { GeneScan-SetT8 }", and the same column of selected cells will fill automatically. Click on "ok". The prompt box clicks "yes". The sample table editing is completed.

In the 'non-running form', clicking a form with the name of the sample form being '8 dye-matrix', clicking 'link to the A tray' (selecting the A tray or the B tray according to actual needs) under a grid on the right side of the interface, wherein the grid of the sample to be detected is changed into light green at the moment, and the sample form is successfully linked. The click runs and the spectrum correction results are shown in fig. 10.

The result shows that the high-quality eight-color fluorescence spectrum calibration reagent is obtained in example 1, the eight-color fluorescence spectrum can be effectively separated, the mass number is not lower than 0.94 and the passing rate reaches 100% based on the domestic GA118-24B, and all the 24 channels pass through the eight-color spectrum calibration, as shown in FIGS. 11-12.

The eight-color fluorescence spectrum correction result and the spectrum distribution thereof completely meet the requirements of eight-color fluorescence detection.

Example 3

The embodiment provides an application of STR detection of an eight-color fluorescence spectrum calibration result.

After the Matrix file is created in embodiment 2, the file obtained by using the fluorescent calibration reagent is used to collect and process the fluorescent signal of the DNA sample to be tested, and the obtained STR typing results are shown in fig. 13.

The results show that: after being calibrated by the eight-color fluorescence calibration reagent, GA118-24B can correctly identify the corresponding eight-color fluorescence composite amplification product, and no fluorescence permeation phenomenon is generated among all fluorescence channels.

The detection result shows that for the standard reference sample, the eight-color fluorescent amplification reagent and the detection platform thereof are adopted to obtain a complete STR typing result, the peak shape is sharp, no site loss phenomenon occurs, the typing result is correct, and the requirements of individual identification and identification are completely met.

Various corresponding changes and modifications can be made by those skilled in the art based on the above technical solutions and concepts, and all such changes and modifications should be included in the protection scope of the present invention.

SEQUENCE LISTING

<110> first institute of police department

BEIJING ZHONGDUN ANMIN ANALYSIS TECHNOLOGY Co.,Ltd.

<120> eight-color fluorescence spectrum calibration reagent and preparation method thereof

<130> 123

<160> 24

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<213> Artificial sequence

<400> 19

gatattaaca gtaactgcct tcatagata 29

<210> 20

<211> 21

<212> DNA

<213> Artificial sequence

<400> 20

ctgaaaccga tttgataacc g 21

<210> 21

<211> 27

<212> DNA

<213> Artificial sequence

<400> 21

tttgtatttc atgtgtacat tcgtatc 27

<210> 22

<211> 22

<212> DNA

<213> Artificial sequence

<400> 22

cgttggctgc atagacgtta tt 22

<210> 23

<211> 21

<212> DNA

<213> Artificial sequence

<400> 23

cattggcctg ttcctccctt a 21

<210> 24

<211> 24

<212> DNA

<213> Artificial sequence

<400> 24

cctacagtaa cgcgaactga ctct 24