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Eight-color fluorescence spectrum calibration reagent and preparation method thereof

A technology of fluorescence spectroscopy and calibration reagents, which is applied in the field of DNA detection, can solve problems such as complex and cumbersome preparation process, affect the quality of spectral calibration reagents, and reduce the mass number, and achieve the effect of simplifying the preparation process, avoiding operation, and easy operation

Pending Publication Date: 2021-12-03
THE FIRST RES INST OF MIN OF PUBLIC SECURITY +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Chinese patent application ZL201110201443.3 discloses a five-color fluorescent matrix standard and its preparation method and special primer composition. The 9947A standard DNA is used as a template and multiple pairs of primers are used for multiplex amplification. A shadow band (Stutter) is generated, resulting in multiple impurity peaks in the fluorescence spectrum calibration reagent, which greatly affects the quality of the spectrum calibration reagent, resulting in a decrease in the mass number
In addition, the preparation of traditional fluorescence spectrum calibration reagents mostly adopts single fluorescent fragment amplification, purification, mixing, and blending methods, and the preparation process is complicated and tedious.

Method used

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  • Eight-color fluorescence spectrum calibration reagent and preparation method thereof
  • Eight-color fluorescence spectrum calibration reagent and preparation method thereof
  • Eight-color fluorescence spectrum calibration reagent and preparation method thereof

Examples

Experimental program
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Embodiment 1

[0038] This embodiment provides a method for preparing an eight-color fluorescence spectrum calibration reagent, and the specific process is as follows:

[0039] S1. Artificially synthesize 8 template DNAs, each template DNA contains binding sites for different primers, and the 8 template DNAs will be connected to the PMD-18T carrier through T / A; the sequence information of the 8 template DNAs is shown as 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.

[0040] S2. Design and determine 8 pairs of fluorescent primers, the sequence information of the 8 pairs of fluorescent primers are 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.

[0041] Table 1

[0042]

[0043...

Embodiment 2

[0066] This embodiment provides a method for generating an eight-color fluorescence calibration file.

[0067] The eight-color fluorescence calibration reagent prepared in Example 1 needs to be tested by a domestic genetic analyzer GA118-24B to generate a corresponding spectral calibration file for the detection of the corresponding eight-color fluorescence-labeled fluorescent PCR products.

[0068] Take 25 μL eight-color fluorescence calibration reagent, mix with 225 μL formamide and mix thoroughly, after denaturation at 95°C for 5 minutes and -20°C for 5 minutes, dispense 10 μL in each of the 24 wells in three rows (A1~H3) of a 96-well plate, Use the GA118-24B genetic analyzer to perform detection according to the operating instructions, and the specific parameters are as follows:

[0069] Select T8 fluorescence channel mode

[0070] The capillary length is 36cm;

[0071] Electrophoresis conditions: temperature is 60°C, gel filling time is 15s, electrophoresis voltage is 1...

Embodiment 3

[0080] This embodiment provides an STR detection application of the calibration result of the eight-color fluorescence spectrum.

[0081] After the Matrix file is established in Example 2, the file obtained by using the fluorescence calibration reagent is used to collect and process the fluorescence signal of the DNA sample to be tested, and the obtained STR typing results are as follows: Figure 13 shown.

[0082] 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 products, and there is no fluorescence penetration phenomenon between each fluorescence channel.

[0083] The test results show that, for the standard reference sample, the eight-color fluorescent amplification reagent and its detection platform are used to obtain a complete STR typing result, and the peak shape is sharp, no site loss occurs, and the typing result is corr...

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Abstract

The invention discloses an eight-color fluorescence spectrum calibration reagent and a preparation method thereof, eight template DNAs (deoxyribonucleic acid) which are artificially designed and synthesized are adopted, each template DNA template comprises a binding site of a pair of primers, and accurate control of fragment length 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 is labeled by eight different fluorescence, multiplex amplification is carried out in a specific reaction system, the fluorescence spectrum calibration reagent containing eight fluorescence products can be obtained at the same time, and the fluorescence spectrum calibration reagent can be directly applied to fluorescence spectrum calibration without mixing and blending monochromatic fluorescence fragments. According to the method, the preparation process flow of the fluorescent standard substance is greatly simplified, and complex and tedious operation is avoided. The preparation method disclosed by the invention can be used for stably obtaining the standard substance and has the characteristics of high efficiency and simplicity and convenience in operation.

Description

technical field [0001] 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 technique [0002] DNA detection technology has become one of the most important technical means in the field of modern forensic science, and it is the most effective individual identification tool in handling various cases, major accidents and natural disasters and other major events. DNA testing equipment and DNA testing reagents are the two core elements for the development of DNA testing. [0003] The most common testing equipment in domestic DNA testing laboratories are mainly 3130, 3130xl, 3500, 3500xl genetic analyzers. Instruments and equipment in the field of international forensic science DNA testing have experienced four-color, five-color to six-color development. [0004] The earliest DNA reagent system used for DNA detection was the TH01, FES\FPS, vWA and F13...

Claims

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Application Information

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IPC IPC(8): C12N15/11G01N21/64C12Q1/6806
CPCC12Q1/6806G01N21/6428G01N2021/6439C12Q2563/107C12Q2527/125
Inventor 姜伯玮张黎明王峰荣海博赵怡鹤张涛金川陈力
Owner THE FIRST RES INST OF MIN OF PUBLIC SECURITY
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