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Kit to detect fetal chromosomal non-integral multiple and method to detect fetal methylated DNA copy number

A non-integer multiple and kit technology, applied in the medical field, can solve the problems of deviation of test results, different sample sizes, different copy numbers, etc., and achieve the effect of improving detection accuracy and balancing errors

Pending Publication Date: 2019-01-04
领航医学科技(深圳)有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Theoretically, these specific differentially methylated nucleic acid fragments can be completely distinguished from maternal and fetal nucleic acid fragments by bisulfite treatment technology, methylation-sensitive restriction endonuclease technology or DNA co-immunoprecipitation technology. Yes, there are differences in the degree of methylation between different fetal specific methylated nucleic acid fragments, so the acquisition efficiency of each fetal methylated nucleic acid fragment is different, even if the same methylated nucleic acid fragment has a different elution efficiency each time, This will result in different sample sizes for each test, and the copy number obtained by digital PCR will also be different, which will eventually lead to deviations in test results

Method used

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  • Kit to detect fetal chromosomal non-integral multiple and method to detect fetal methylated DNA copy number
  • Kit to detect fetal chromosomal non-integral multiple and method to detect fetal methylated DNA copy number
  • Kit to detect fetal chromosomal non-integral multiple and method to detect fetal methylated DNA copy number

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Experimental program
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Effect test

Embodiment 1

[0094] Embodiment 1: The present invention detects the kit of fetal chromosome non-integer multiple (21-trisomy syndrome)

[0095] 1. Maternal plasma cell-free nucleic acid extraction reagent

[0096] Lysis solution: 7-9M guanidine hydrochloride, 2-6M guanidine isothiocyanate, 10-50mM EDTA and 20-100mM Tris-HCl;

[0097] Binding solution: 70% isopropanol and 30% ethanol;

[0098] Rinsing solution A: isopropanol: ethanol: lysate = 2:3:5;

[0099] Rinse solution B: 70%-90% ethanol;

[0100] Eluent: TE solution, 10mM Tris-HCl, 1mM EDTA, pH=8.0;

[0101] Magnetic beads: surface-modified (hydroxyl, carboxyl, etc.) ferric oxide;

[0102] and proteinase K;

[0103] 2. Methylated DNA co-immunoprecipitation reagent

[0104] SimpleDIP Hydroxy / Methylated DNA IP (hMeDIP) Kit produced by CST, the article number is #95176𒰵;

[0105] 3. Digital PCR detection reagent

[0106] Chip: After laser etching, about 20,000 micro-reaction units are formed on the silicon wafer;

[0107] PCR m...

Embodiment 2

[0112] Example 2: Optimization of optimal antibody amount for co-immunoprecipitation

[0113]In order to ensure the specific binding of the 5-mc antibody to the fetal methylated nucleic acid fragment, the present invention sets four antibody additions of 1000ng, 500ng, 200ng and 50ng, and takes the plasma (negative sample) of a normal non-pregnant adult female as a sample, The target gene is the target gene and target gene in Table 1. The results show that when the antibody addition amount is 50ng, the detection copy number of the target gene is close to 0, so 50ng antibody addition amount is selected during co-immunoprecipitation.

Embodiment 3

[0114] Embodiment 3: Signal amplification test of different target fragment detection

[0115] The present invention is tested with 3 target segments on human chromosome 13, 3 target segments on human chromosome 18 and 2 target segments on human chromosome 21, and these segments are all related to non-integer multiple chromosome diseases Differentially methylated DNA fragments, as follows:

[0116] chr13:97356672-97356757, abbreviated as 13MBNL2;

[0117] chr13:95247607-95247759, abbreviated as 13ABCC4;

[0118] chr13:21140893-21140975, abbreviated as 13SAP18;

[0119] chr18: 25168268-25168357, abbreviated as 18EHZF;

[0120] chr18:75202144-75202225, abbreviated as 18ZADH2;

[0121] chr18:659516-659663, abbreviated as 18TYms;

[0122] chr21: 43669944-43670035, abbreviated as 21RRP1B;

[0123] chr21: 37225160-37225290, abbreviated as 21DSCR3;

[0124] Primers and probes were designed according to the above fragments, and different target fragments on the same chromosome ...

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Abstract

The invention relates to the technical field of medicine and discloses a kit to detect fetal chromosomal non-integral multiple and a method to detect fetal methylated DNA copy number. The kit includesa maternal plasma free nucleic acid extracting agent, a specific nucleic fragment enrichment kit, a digital PCR (polymerase chain reaction) detection reagent and a contribution coefficient table. Contribution coefficients of methylated differential nucleic fragments of chromosomes all related to chromosomal non-integral multiple are set; the contribution coefficients help eliminate different influences upon precipitating efficiency caused by each co-immunoprecipitation; a noninvasive detection method for fetal chromosomal anomalies is completed; detection results are closer to real results; no dependence is posed on the ratio of fetal nucleic acid in a maternal plasma sample; the problem that super-early screening faces low fetal ratio is solved to great extent.

Description

technical field [0001] The invention relates to the field of medical technology, in particular to a kit for detecting non-integer multiples of fetal chromosomes and a method for detecting the copy number of fetal methylated DNA. Background technique [0002] Prenatal diagnosis is an effective detection method to prevent the birth of defective babies, and it has received extensive attention. The specific inspection methods are divided into invasive and noninvasive. Invasive detection methods are mainly fetal chorionic villus sampling and amniocentesis, etc. This detection method has the risk of causing fetal miscarriage, so its clinical application has certain limitations. In recent years, non-invasive prenatal diagnosis has attracted more and more attention. [0003] The discovery of fetal cell-free DNA in the peripheral blood of pregnant women provides an important research basis for non-invasive prenatal diagnosis. Studies have shown that the proportion of fetal free DNA...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/6851
CPCC12Q1/6851C12Q1/6883C12Q2600/166C12Q2600/154C12Q2531/113C12Q2537/16C12Q2537/165C12Q2545/113
Inventor 朱留伟
Owner 领航医学科技(深圳)有限公司