Primers, probes, kit and method for noninvasive prenatal diagnosis of Bart's hydrops syndrome based on droplet digital PCR

A prenatal diagnosis, droplet technology, applied in biochemical equipment and methods, microbial determination/examination, DNA/RNA fragments, etc. Control methods and other issues, to achieve the effect of mature detection platform technology, high sensitivity, and good detection repeatability

Active Publication Date: 2019-11-12
GUANGDONG WOMEN & CHILDREN HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

High-throughput sequencing can completely detect the fetal genome in the plasma of pregnant women, and identify fetal mutation sites with the help of bioinformatics methods. However, the experimental cycle is long, the experimental cost is high, and it is easily affected by factors such as library construction and fetal concentration, so it is difficult to popularize
Fluorescent quantitative PCR has poor reproducibility in detecting trace amounts of DNA, and it is difficult to effectively detect trace changes in fetal genome content, lacking quality control methods

Method used

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  • Primers, probes, kit and method for noninvasive prenatal diagnosis of Bart's hydrops syndrome based on droplet digital PCR
  • Primers, probes, kit and method for noninvasive prenatal diagnosis of Bart's hydrops syndrome based on droplet digital PCR
  • Primers, probes, kit and method for noninvasive prenatal diagnosis of Bart's hydrops syndrome based on droplet digital PCR

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0060] 1. The primers and probes for the non-invasive prenatal detection of Pap edema fetuses based on the droplet digital PCR platform are as follows (including 2 pairs of primers and 1 pair of probes):

[0061] α-globin gene cluster quantitative target gene upstream primers are:

[0062] 5'-ACTTCCTCAGTGGCAAAC-3' (SEQ ID NO: 1);

[0063] α-globin gene cluster quantitative target gene downstream primers are:

[0064] 5'-TAAACCTGCTCTGTCTG-3' (SEQ ID NO: 2);

[0065] The α-globin gene cluster quantitative target gene probes are:

[0066] 5'-FAM-ACTGAGGACTTCCTCCCGACCTCAT-MGB-3' (SEQ ID NO: 3); wherein, FAM represents carboxyfluorescein labeling; MGB represents Minor Groove Binder.

[0067] The upstream primers of the internal reference gene ALB are:

[0068] 5'-TGTTGCATGAGAAAACGCCA-3' (SEQ ID NO: 4);

[0069] The downstream primers of the internal reference gene ALB are:

[0070] 5'-TTCATCGACTTCCAGAGCT-3' (SEQ ID NO: 5);

[0071] The probe of the internal reference gene AL...

Embodiment 2

[0082] (1) Extraction of plasma from pregnant women: Take 10 mL of peripheral blood from pregnant women, store and transport it in EDTA tubes, separate the plasma within 6 hours after blood drawing, and centrifuge at 1600×g for 10 minutes; take the supernatant and centrifuge at 16000×g for 10 minutes; take the supernatant The solution was dispensed into 2mL centrifuge tubes and stored in a -30°C or -80°C refrigerator for a long time. Take out each tube of plasma once to avoid repeated freezing and thawing;

[0083] (2) Extract free DNA from the plasma of pregnant women; use no less than 1200 μL (about 1200 μL ~ 2000 μL) of plasma. Extraction was performed using HiPure circulating DNA acid Midi Kit (Magen) (or MagPure Mini Kit or HiPure circulating DNA LQ Kit (Magen)). Divide the plasma into multiple tubes for extraction according to the actual amount of plasma used, use TE eluent, and then combine the eluents extracted from the same plasma after elution. Note that the combine...

Embodiment 3

[0107] 1. Detection of samples with known genotypes

[0108] The gDNA whose genotype is -- / --, that is, Bartholin's edema fetus, is mixed with the known genotype -- / αα (that is, Southeast Asia α-thalassemia genotype), gDNA with genotype -- / -- was extracted from fetal placental villi samples, and gDNA with genotype -- / αα was extracted from peripheral blood of pregnant women, both using Qiamp DNA Blood Mini kit (article number: 51106, Qiagen, USA). Before mixing, the genotypes of -- / -- and -- / αα have been diluted to the same concentration, and after mixing, the concentration of each concentration gradient sample is diluted to 10ng / μL, 5ng / μL, 2ng / μL, using the reagent of the present invention A 25 μL total volume reaction system was used for detection (method refer to Example 2). The result is as image 3 shown.

[0109] 2. The sensitivity and specificity of the kit

[0110] The plasma (1.2mL each) separated from the peripheral blood of 25 pregnant women was drawn separatel...

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Abstract

The invention discloses primers, probes, a kit and a method for noninvasive prenatal diagnosis of Bart's hydrops syndrome based on droplet digital PCR. The primers and the probes comprise upstream anddownstream primers of an alpha globin gene cluster quantitative target gene and alpha globin gene cluster quantitative target gene probes, with nucleotide sequences shown in SEQ ID NO:1-3 respectively. The primers and probes also comprise upstream and downstream primers and probes of a reference gene ALB, with nucleotide sequences shown in SEQ ID NO:4-6 respectively. The primers and the probes have the advantages of rapidness, high throughput, high sensitivity and lower detection cost, can be prepared into the kit for noninvasive prenatal diagnosis of Bart's hydrops syndrome and are particularly applicable to prenatal diagnosis of carriers whose parents are both Southeast Asia deletion.

Description

technical field [0001] The invention relates to the field of non-invasive prenatal diagnosis of monogenic diseases, in particular to a primer, probe, kit and method for non-invasive prenatal diagnosis of Pap edema fetuses based on droplet digital PCR. Background technique [0002] α-thalassemia is caused by the abnormality of the α-globin gene, which leads to the failure of the synthesis of related globin chains. α-thalassemia is one of the most common types of thalassemia in China, and the highest level is in Guangdong and Guangxi. According to the number of copies of the functional α-globin gene, it can be divided into static thalassemia, standard thalassemia, HbH disease, and Bartholin's edema, with 3, 2, 1, and 0 copies of the α-globin gene, respectively. Divided into α according to the functional globin gene on a single chain + and alpha 0 Thalassemia, alpha + Common types of thalassemia include right-sided deletions (-α 3.7 ) and deletions on the left (-α 4.2 ) tw...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12N15/11C12Q1/6851
CPCC12Q1/6883C12Q1/6851C12Q2600/156C12Q2531/113C12Q2563/159
Inventor 尹爱华何天文陈汉彪张亮王梓铭
Owner GUANGDONG WOMEN & CHILDREN HOSPITAL
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