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Organ transplantation ddcfDNA detection reagent and method

A technology of reagents and diagnostic methods, applied in the field of medical molecular biology, can solve the problems of restricting wide application, troublesome and expensive, obvious limitations, etc., and achieve the effect of accurate and sensitive detection results

Active Publication Date: 2021-03-09
北京迈基诺基因科技股份有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The methods for detecting ddfDNA are as follows: First, in female recipients who have received male organs, detection of the SRY or TSRY region on the Y chromosome is the simplest and most direct method, but due to obvious limitations, it also limits its wide application The second is to detect polymorphic genes or regions (such as HLA) among individuals by means of qPCR, ddPCR, MPSS, etc., so as to distinguish the cfDNA of the recipient and the donor. Typing, laborious and expensive

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  • Organ transplantation ddcfDNA detection reagent and method
  • Organ transplantation ddcfDNA detection reagent and method
  • Organ transplantation ddcfDNA detection reagent and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Example 1 Establishment of detection method for free DNA concentration in recipient plasma

[0031] The establishment of a standard curve

[0032] 1 Primer design and selection: ALU sequence is a rich repeat sequence in the human genome, accounting for about 5%-10% of the genome, and is scattered throughout the genome with an average interval of 4kb. The present invention utilizes the ALU repeat sequence to design 81bp, 115bp primers to To quantify the amount of cfDNA in plasma, the sequence is shown in Table 1

[0033] Table 1 Primer Sequence

[0034] Primer name Primer sequence Seq NO ALU81-F 5'-CCTGAGGTCAGGAGTTCGAG-3' 1 ALU81-R 5'-GCCCCGGCTAATTTTTGTAT-3' 2 ALU115-F 5'-CCTGAGGTCAGGAGTTCGAG-3' 3 ALU115-R 5'-CCCGAGTAGCTGGGATTACA-3' 4

[0035] 2 Dilute the Coriell institute NA12878 genomic standard gDNA to 0.1pg / µL, 1.0pg / µL, 10pg / µL, 100pg / µL;

[0036] 3 Prepare the reaction body according to Table 2 and the quantitati...

Embodiment 2

[0056] Example 2. Establishment of a method for detecting the relative content of ddcfDNA in recipient plasma

[0057] one-probe design

[0058] Obtain the literature reports from UCSC and the sequences near 441 polymorphic SNP sites in the Mikino database. After removing the repeated sequences, intercept the 78bp sequence from the first base as the probe, and move back n again. Base, intercept the 78bp sequence as a probe until the last 78bp.

[0059] Table 4 (some SNPs)

[0060] rs chromosome Location rs chromosome Location rs1490413 chr1 4367323 rs3764570 Chr19 8528277 rs3740199 chr10 128019025 rs528557 chr20 3651742 rs10488710 chr11 115207176 rs3205187 Chr22 31491295 rs10831567 chr11 11292700 rs743616 Chr22 51064039 rs1169289 chr12 121416622 rs13071423 chr3 2140378 rs12825673 chr12 569945 rs6835017 chr4 20182928 rs4530059 chr14 104769149 rs16870629 chr5 1015063 rs2306049 c...

Embodiment 3

[0098] Example 3 Calculation of ddcfDNA absolute value score in recipient plasma

[0099] Calculation of the absolute value score of ddcfDNA in the recipient plasma: the absolute content obtained in Example 1 and the relative content in Table 6 in Example 2 are comprehensively calculated: the total amount of cfDNA × the relative content to obtain an absolute value score, which is verified by a large number of clinical samples. It is found that the absolute value score greater than 0.2 indicates that the sample to be tested is a rejection individual (the clinical diagnosis of organ transplant rejection is based on the pathological diagnosis of transplanted kidney biopsy (according to the internationally uniform Banff standard)).

[0100] Table 7

[0101] sample number Algorithm 1 Relative Value Scoring Algorithm 1 detection result Algorithm 2 Relative Value Scoring Algorithm 2 detection results Algorithm 3 Relative Value Scoring Algorithm 3 detection resul...

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Abstract

The invention provides a non-diagnostic method for quantifying cfDNA by utilizing an ALU repetitive sequence and a ddcfDNA relative content detection method. The obtained data can be used for detecting organ transplantation rejection response.

Description

technical field [0001] The invention belongs to the field of medical molecular biology, in particular to a reagent and method for detecting Donor-Derived Cell-Free DNA in blood plasma or urine after organ transplantation. Background technique [0002] Organ transplantation is an effective treatment for advanced organ diseases, but the success of organ transplantation and long survival time requires many "hurdles", including immune tolerance and rejection, infection, and adaptation to new organs. Rejection and infection are common complications that affect the quality of life and life safety of transplant patients. The traditional tools for diagnosing organ transplant rejection are invasive biopsy and serum creatinine detection, which can evaluate the glomerular filtration rate, but the sensitivity and specificity for distinguishing transplanted kidney injury from acute and chronic failure are not high. Puncture biopsy is the gold standard for detection of transplant rejecti...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/6851G16B40/00
CPCC12Q1/6883C12Q1/6851G16B40/00C12Q2600/156C12Q2600/118C12Q2531/113C12Q2563/107C12Q2545/114
Inventor 伍建姬晓雯王海丽刘娜韩路
Owner 北京迈基诺基因科技股份有限公司
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