Method and kit for detecting pathogenic gene of fetal thalassemia

A technology for thalassemia and disease-causing genes, applied in biochemical equipment and methods, microbial determination/inspection, etc., can solve the problems of false negative PCR and the inability to further determine the existence of maternal mutations.

Active Publication Date: 2022-03-11
GUANGZHOU DARUI BIOTECH +1
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, these methods are based on single-site PCR technology. Due to the low content of plasma DNA and the characteristics of high fragmentation, it is likely to cause false negatives in PCR.
In addition, these methods can only detect the presence of specific paternal mutations that are different from the mother, but cannot further confirm the presence of maternal mutations

Method used

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  • Method and kit for detecting pathogenic gene of fetal thalassemia
  • Method and kit for detecting pathogenic gene of fetal thalassemia
  • Method and kit for detecting pathogenic gene of fetal thalassemia

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Construction of embodiment 1 haplotype (i.e. haploid genotype)

[0045] (1) Thalassemia primer and probe design

[0046] Design region for α-thalassemia (chr16:60022-2233661): downstream (10Kb) + HBA gene + upstream (10Kb), and design region for β-thalassemia (chr11:3236690-7177304): downstream (10Kb) + HBB gene + upstream (10Kp), and then use the dbSNP database to screen out 195 α-thalassemia SNP sites and 275 β-thalassemia SNP sites. Among them, the SNP site screening conditions: the SNP site has MAF>=20% at the same time in the Thousand Genomes Chinese Southerners and Northerners database; the 100bp sequence above and below the SNP site is a specific region and has no homology on the genome; and 45 %

Embodiment 3

[0072] Embodiment 3 maternal genetic situation analysis

[0073] Based on the analysis of paternal inheritance, the analysis of maternal inheritance was carried out. Select the locus (MN) whose genotype is homozygous father (MM) and heterozygous mother (MN), and determine whether the maternal pathogenic haplotype is inherited according to RHDO-SPRT, wherein the SPRT curve is calculated according to the following formula:

[0074]

[0075] in,

[0076] Upper boundary and Lower boundary refer to the upper bound and the lower bound respectively. In this detection method, the inventor defines haplotype 1 as the upper bound, and defines haplotype 2 as the lower bound. Use the SPRT algorithm to calculate the sum of the sum of the variation frequencies of the selected sites corresponding to the depth. If the screened loci are all between the upper and lower bounds, then it is impossible to tell which haplotype the fetus has inherited from the mother; if one or more of the acc...

Embodiment 4

[0078] Example 4 Test results of 5 thalassemia families

[0079] (1) Thalassemia test results

[0080] Families 1-5 were detected by the method of Example 2, and the haplotype construction results of families 1-5 are shown in Table 3-7. According to Mendel's law of inheritance, combined with family sample phenotype and known mutation type information, the pathogenic mutation is associated with the haplotype (for example: the father's genotype is MN, the mother's genotype is MN, and the child's genotype is NN (M is the disease-causing locus, N is the normal locus. M and N represent the bases A, T, C, G), and the child has inherited the father's haplotype 1 and the mother's haplotype 1 respectively, then The father's pathogenic locus M is on haplotype 2, and the mother's pathogenic locus M is on haplotype 2). Table 8 shows the results of the association of pathogenic loci in the father and mother of families 1-5 with haplotypes.

[0081] Table 3 Haplotype construction results...

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Abstract

The invention discloses a method for detecting fetal thalassemia-causing genes, comprising the following steps: (1) screening SNP sites, the SNP sites are used to design primer pools for amplifying genomic thalassemia genes and capturing free DNA in plasma of pregnant women The probe of thalassemia gene; (2) extract the free DNA in the plasma of pregnant women and the whole blood genomic DNA of three born compatriots and construct the corresponding DNA library for template preparation and enrichment; (3) to step (2) Sequencing of cell-free DNA and whole blood genomic DNA library; (4) Construct the haploid genotype of the SNP site on the thalassemia gene, combine the sequencing information of cell-free DNA and whole blood genomic DNA library, and analyze paternal inheritance and maternal Source genetic conditions to determine the corresponding genotype of the fetal SNP locus. The present invention realizes the non-invasive prenatal detection of thalassemia through target region capture combined with high-throughput sequencing technology; the required sample size is small; the method can realize the detection of fetal maternal gene mutations on the basis of detection of paternal mutations detection.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a method and a kit for detecting fetal thalassemia-causing genes. Background technique [0002] Thalassemia (referred to as thalassemia) is one of the high-incidence genetic diseases in the world. It is due to the mutation or deletion of human α, β-globin gene, which leads to the imbalance of α, β-globin peptide chain synthesis rate, which causes hemolysis. sexual anemia. The two common types of thalassemia are α-thalassemia and β-thalassemia, α-thalassemia-related genes are HBA2 and HBA1, and β-thalassemia-related genes are HBB. Thalassemia is concentrated in tropical and subtropical regions, mostly in Mediterranean countries, followed by the Middle East, India, Pakistan, Southeast Asia, southern China and North Africa. The United States is an immigrant country, and its incidence is also relatively high. The provinces south of the Yangtze River in my country are high-in...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6869C12Q1/6883
CPCC12Q1/6869C12Q1/6883C12Q2600/156C12Q2531/113C12Q2537/143C12Q2525/191C12Q2535/122C12Q2537/165
Inventor 吴英松李明范冬梅林圣杨旭侯荣基
Owner GUANGZHOU DARUI BIOTECH
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