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NGS database creating primer pool used for amplifying multiple targets in cfDNA sample and application

A primer pool and universal primer technology, applied in DNA/RNA fragmentation, recombinant DNA technology, microbial determination/inspection, etc., can solve the problem of difficult implementation of multiplex PCR technology, and achieve low non-specific amplification and easy amplification. Effect

Active Publication Date: 2017-10-17
广州万德基因医学科技有限公司
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AI Technical Summary

Problems solved by technology

Large format multiplex PCR technology is very difficult to achieve

Method used

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  • NGS database creating primer pool used for amplifying multiple targets in cfDNA sample and application
  • NGS database creating primer pool used for amplifying multiple targets in cfDNA sample and application
  • NGS database creating primer pool used for amplifying multiple targets in cfDNA sample and application

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Embodiment 1

[0053] Example 1: Detection of fetal chromosomal aneuploidy using peripheral blood of pregnant women. It is used to verify the application of the present invention in the field of NIPT (non-invasive prenatal testing), which can correctly identify multiple chromosomal aneuploidies selected from trisomy 21, trisomy 13, trisomy 18 and monosomy X positive sample.

[0054] 1. Blood sample collection and processing

[0055] 1) 5ml of peripheral blood was collected intravenously from 275 pregnant women (gestational weeks over 12 weeks).

[0056] 2) Plasma separation: the blood collection tube was centrifuged at 1600 g for 10 min. Take supernatant plasma and transfer to EP tube. Centrifuge the above-mentioned EP tube at 16000 g for 10 min, point the tip of the pipette to the non-white blood cell precipitate, and absorb the supernatant. Proceed to the next step immediately or store in a -80°C refrigerator.

[0057] 3) Use Qiagen plasma DNA extraction kit to extract cfDNA in plasma...

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Abstract

The invention discloses an NGS database creating primer pool used for amplifying multiple targets in a cfDNA sample and application. Each primer in the primer pool is composed of a universal primer segment and a specific primer segment, wherein the universal primer segment is connected with a terminal 5' of a specific primer, a (N)k sequence used for adjusting a primer Tm is connected between the universal primer segment and the specific primer segment of at least one primer, and difference between Tm values of the primers is not more than 10 DEG C. The primers in the primer pool have almost the same Tm, non-specific amplification is lower, further amplification is easy, and the number of covering layers in a detection target area can be reached conveniently. The method has the advantages that thousands of PCR can be carried out in the same system at the same time, and the limit that the conventional multiple PCR generally does not exceed 100 is broken.

Description

technical field [0001] The invention relates to a set of primer pools for multiplex PCR and applications thereof, in particular to NGS library construction primer pools for amplifying multiple cfDNA targets in samples and applications thereof. Background technique [0002] WGS whole-genome sequencing can obtain structural variations such as mutations, insertions, deletions, and copy numbers of the entire genome. However, due to the huge amount of whole genome data, taking 30X sequencing as an example, the whole human genome will generate more than 90G of sequencing data. The low mutation frequency related to tumors, and the sequencing of insertions, deletions, and copy numbers require at least 5000X coverage, and whole-genome sequencing will generate more than 15T of sequencing data. In particular, the tumor-associated cell-free DNA in body fluid samples, or the cell-free DNA from the fetus in the peripheral blood of pregnant women is extremely low, and the sequencing volum...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6848C12Q2537/143C12Q2531/113
Inventor 陈梦麟黄凯铃骆颖筠
Owner 广州万德基因医学科技有限公司
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