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Method for preparing cfDNA library qPCR quantification standard substance

A quantitative standard product and library technology, applied in biochemical equipment and methods, microbial measurement/testing, etc., can solve problems such as tailing peaks, not well representing the real situation of cfDNA, and expensive prices

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  • Description
  • Claims
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Problems solved by technology

The common feature of these kits is that according to the known concentration of the standard, the relative quantification of the library is carried out by using the SYBR fluorescent dye method. The fragment length of the general standard is 399-452bp, which is not specifically for the cfDNA library, and these commercial reagents The price of the box is relatively expensive, especially the price of the standard product, which is about 100-300 yuan / time, and only 25 libraries can be quantified at a time
Patent 201510442279.3 discloses a patent for quantitative standards of cfDNA library, but this patent uses semiconductor sequencing method, and uses the method of breaking the genome into 160-200bp fragments to make standards, but the breaking products are not concentrated , there is a tailing peak, which cannot well represent the real situation of cfDNA

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  • Method for preparing cfDNA library qPCR quantification standard substance
  • Method for preparing cfDNA library qPCR quantification standard substance
  • Method for preparing cfDNA library qPCR quantification standard substance

Examples

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Effect test

Embodiment 1

[0040] Example 1 Extraction of cfDNA

[0041] Use the QIAamp Circulating Nucleic Acid Kit (Qiagen) to extract cfDNA from 10ml of peripheral blood. The specific steps are as follows:

[0042] 1. Use K2EDTA 10ml vacuum blood collection tube (BD) to collect 10ml of peripheral blood from healthy people, and separate plasma within 4 hours.

[0043] 2. Plasma separation step: centrifuge at 1600g for 10min, take the supernatant, and be careful not to suck the white blood cells in the middle layer (after this centrifugation, the supernatant is plasma, the middle layer is white blood cells and platelets, and the lower layer is red blood cells); the supernatant Centrifuge at 16,000g for 10 minutes to remove residual cells, and do not absorb the white blood cells at the bottom; the separated plasma is stored in separate tubes at around -80°C.

[0044] 3. Take out the plasma from the -80°C refrigerator, thaw it on ice, centrifuge at 16000g for 10min, and transfer the supernatant.

[004...

Embodiment 2

[0050] Example 2 cfDNA library construction

[0051] Use the Kapa Hyper Prep Kit Illumina kit for cfDNA library construction, the specific steps are as follows:

[0052] 1. End repair and add "A"

[0053] First prepare 7 μl of end repair plus A base buffer and 3 μl of end repair plus A base enzyme mixture. After mixing, gently pipette 10 times with a gun to mix well, detach lightly, and place on ice. Then add the cfDNA sample extracted in Example 1 to 50 μl with ddH2O, add it to the PCR reaction tube, and incubate according to the following conditions: 20°C, 30min; 65°C, 30min; 4°C, keep.

[0054] 2. Add Index connector

[0055] First prepare a reaction solution containing 30 μl of ion buffer and 10 μl of DNA ligase, gently pipette 10 times with a gun to mix, lightly detach, and add to a reaction tube of 60 μl of the reaction product of end repair and A base addition. Then add Index linker reaction solution (10 μM) and PCR grade water to the reaction tube to a final volume ...

Embodiment 3

[0066] Example 3 Accurate Quantification of cfDNA Library

[0067] Using the third-generation digital PCR QX200 Droplet Digital PCR system and supporting reagent ddPCR TM The Library Quantification Kit for Illumina performs precise absolute quantification of cfDNA libraries.

[0068] 1. First, according to Table 1, dilute the cfDNA library with TE buffer, and prepare a mixture containing 10 μl of 2XddPCR probe mixture, 1 μl of 20X ddPCR library quantitative reaction solution and 5 μl of DNase and RNase-free water liquid.

[0069] Table 1 Gradient dilution ratio table

[0070]

[0071] 2. Set the above dilution ratio to 10 -6 、10 -7 、10 -8 、10 -10 The samples were detected in a 96-well plate, and 16 μl of the mixed solution and 4 μl of the diluted library were added to each reaction well, and 3 replicates were performed, and 3 replicates of NTC were added to the same plate. Then parafilm, mix well and centrifuge briefly.

[0072] 3. Operate according to the instructi...

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Abstract

The invention relates to a method for preparing a cfDNA library qPCR quantification standard substance. The method comprises the steps of extracting cfDNA, wherein the length of cfDNA is 160-200 bp, and the concentration of cfDNA is 0.3-1 ng / [mu]l; establishing a cfDNA library by means of cfDNA, wherein the fragment size of the cfDNA library is 280-320 bp, and the concentration of the cfDNA library is 100-150 ng / [mu]l; conducting precise quantification on the cfDNA library by means of the third generation of digital PCR QX200Droplet Digital PCR system, so as to obtain the cfDNA library qPCR quantification standard substance, wherein the concentration of the cfDNA library qPCR quantification standard substance is 10-100 pM, and the length of the cfDNA library qPCR quantification standard substance is 280-320 bp. The qPCR standard substance is prepared specially for the cfDNA high-throughput sequencing library, so as to improve the precision of cfDNA library quantification and reduce the data size error of high-throughput sequencing.

Description

technical field [0001] The invention relates to the technical field of biological products, in particular to a method for preparing a cfDNA library qPCR quantitative standard. Background technique [0002] Since 454 Life Sciences (the company was officially acquired by Roche in 2007) launched the 454FLX pyrosequencing platform (454 FLX pyrosequencing platform) in 2005, high-throughput sequencing (also known as: next-generation sequencing or next-generation sequencing) has emerged . Immediately afterwards, in 2006, the Solexa Genome Analyzer platform was launched by Illumina in the United States, and in 2007, ABI launched the self-developed SOLiD sequencer (ABISOLiD sequencer). These three sequencing platforms became the core of the high-throughput sequencing platform main representative. [0003] With the continuous and rapid development of high-throughput sequencing, ABI SOLID sequencer and Rohce 454 sequencer have been eliminated from the market, and new sequencing platf...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6851C12Q2535/122C12Q2545/114
Inventor 胡妮徐健唐元华
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