SNP molecular marker for detecting heterologous cfdna, detection method and application

A molecular labeling and heterologous technology, applied in the direction of DNA / RNA fragments, recombinant DNA technology, biochemical equipment and methods, etc., can solve the problem of providing individualized drug guidance for patients who cannot be organ transplanted, lack of detection, and inability to specifically extract cfDNA signals and other problems, to achieve the effect of wide detection range, high detection accuracy and high gene polymorphism

Active Publication Date: 2020-03-17
上海奥根诊断技术有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Therefore, the first technical problem to be solved by the present invention is to overcome the lack of detection of genes related to renal transplantation rejection in the prior art and the inability to provide individualized drug guidance for organ transplantation patients, thereby providing a drug for kidney transplantation SNP molecular markers for early detection of acute rejection and drug metabolism genes with high sensitivity and high specificity, as well as their probes, chips, and kits
[0008] The second technical problem to be solved by the present invention is to overcome the inability to specifically extract cfDNA signals to distinguish deviations and sequencing errors in the prior art, thereby providing a single-molecule labeling adapter to realize single-molecule labeling of cfDNA

Method used

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  • SNP molecular marker for detecting heterologous cfdna, detection method and application
  • SNP molecular marker for detecting heterologous cfdna, detection method and application
  • SNP molecular marker for detecting heterologous cfdna, detection method and application

Examples

Experimental program
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Effect test

Embodiment 1

[0064] Example 1 SNP site screening

[0065] Select the dbSNP (data base SNP, https: / / www.ncbi.nlm.nih.gov / snp / ) database and the Chinese population database (stored in Suzhou Renren Gene Technology Co., Ltd.), the Chinese population database is based on 3000 cases of Chinese The catalog of genetic polymorphism sites constructed from the whole genome sample information, such as figure 1 As shown, there are some differential frequency information between the Chinese population database and the Hapmap database (http: / / www.hapmap.org), which represent the differential genetic loci in the Chinese population, enabling the Chinese population database to more accurately present the Chinese population genetic polymorphism information.

[0066] Sites with a minor allele frequency (MAF) close to 0.5 were screened from the above two databases, and 5754 target SNP sites shown in Table 1 were obtained.

[0067] The SNP sites of the drug metabolism genes, such as CYP3A4, CYP3A5 and CYP2D6...

Embodiment 2

[0094] Example 2 probe design and chip synthesis

[0095] 1. Probe Design

[0096]According to the coordinate position of the SNP site shown in Table 1 and Table 2 on the genome, the SNP site is used as the coordinate starting point, and the base sequence extending 80 bp bases to the 5' end is used as the sequence information to design the upstream probe. The needle does not include the SNP site sequence; with the SNP site as the coordinate starting point, the base sequence extending 80 bp bases to the 3' end is used as the sequence information to design the downstream probe, the downstream probe does not include the SNP site sequence; the rs1036431 position As an example, intercept the 80 bp sequences upstream and downstream of the SNP to obtain the upstream probe (GGCTTTTGAGCGCAGTTAACTTTTGGGCTGAAGGAAAATGGACATGCAAGCAGGAGGGAGAATCCTAGTTAGACTCCTCG) and the downstream probe (TTTTCACAAGTCATTGTCGATGTCACTGTCAGTGTAGTGGCCTTCAAGCTATAGGGAGCCACTCCTAATGAGCAACATG) respectively. The bidire...

Embodiment 3

[0099] Example 3 Preparation of Unimolecular Labeling Adapter

[0100] Single-molecule marker adapters (synthesized by Sangon Bioengineering (Shanghai) Co., Ltd.) include adapters and specific molecular tags, wherein the adapters include 5'-adapters in the forward sequence and 3'-adapters in the reverse sequence; such as figure 2 with image 3 As shown, the 5'-linker is selected from the 5'-linker-1 shown in SEQ ID NO.1 or the 5'-linker-2 shown in SEQ ID NO.2, and the 3'-linker sequence is selected from SEQ ID 3'-linker-1 shown in NO.3 or 3'-linker-2 shown in SEQ ID NO.4; 5'-linker-2 is a partial sequence of the 3' end of 5'-linker-1, 3 '-Linker-2 is a partial sequence of the 5' end of 3'-Linker-1; as figure 2 As shown, a Y-shaped single-molecule marker adapter-1 is composed of 5'-adapter-1, 3'-adapter-1 and specific molecular tags, such as image 3 As shown, 5'-linker-2, 3'-linker-2 and specific molecular tags constitute a Y-shaped unimolecular marker linker-2; specific ...

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Abstract

The invention discloses an SNP molecular marker for detecting heterogenous cfDNA, a detecting probe based on SNP molecular marker design and a chip, a detecting device for heterogenous cfDNA, a kit and a detecting method. The heterogenous cfDNA in a receptor after kidney transplantation is quantified by the detecting chip and the detecting device, a basis is provided for detection of kidney transplantation rejection, noninvasive early detection with high sensitivity and high specificity on kidney transplantation is realized, and a kidney transplantation patient can be monitored continuously. An SNP molecular marker comprises an SNP site of a drug metabolizing gene, mutation information of a medication gene is provided for a kidney transplantation rejection patient, and individualized medication is realized. The invention further provides a single molecular marker joint. The single molecular marker joint is used for constructing a sequencing library to effectively remove repeated data and error introduced randomly in sequencing and PCR processes, the sequencing background noises are reduced, and the detection accuracy is improved.

Description

technical field [0001] The invention belongs to the technical field of gene detection, and in particular relates to a SNP molecular marker for detecting heterologous cfDNA, a probe, a chip, a kit, a method, a detection device and an application thereof. Background technique [0002] Kidney transplantation is the preferred method for clinical treatment of end-stage renal disease, and the most important reason affecting the survival rate of patients after kidney transplantation is the occurrence of rejection. Transplant rejection is divided into hyperacute, acute, borderline acute, subclinical acute, or chronic. Acute rejection (AR) is currently the main dangerous complication after kidney transplantation, and it is also the cause of chronic rejection and graft failure. most important risk factor. Early diagnosis and timely treatment of AR can effectively reduce the pathological damage of transplanted organs and prolong the survival time of transplanted organs. [0003] The ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6883C12Q1/6869C12N15/11C12M1/34
CPCC12Q1/6869C12Q1/6876C12Q2600/106C12Q2600/156C12Q2535/122
Inventor 杨功达蒋廷亚曾丰波胡秀弟黎小锋陈雅文刘磊周阳
Owner 上海奥根诊断技术有限公司
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