Kit for non-invasive prenatal diagnosis of fetal trisomy in pregnant women and method of use thereof

A technology for trisomy syndrome and prenatal diagnosis, applied in biochemical equipment and methods, microbial determination/examination, etc., can solve the problem of inability to achieve efficient, accurate, fast, and simple detection requirements, and the difficulty of single marker detection. requirements, multi-marker combined detection methods are cumbersome and other problems, to achieve the effect of being suitable for popularization, avoiding false negative and false positive amplification, and using simple and fast methods

Active Publication Date: 2021-01-15
青岛千卓分子生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Existing amniotic fluid, umbilical cord blood puncture or chorionic villus sampling detection is highly accurate, but it is easy to cause great pain to pregnant women
Among the non-invasive detection methods, ultrasound detection has low accuracy and is suitable for auxiliary screening; in serological detection, it is difficult for a single marker to meet the detection requirements, while the multi-marker combined detection method is cumbersome, serological detection is complicated, and the accuracy is limited
The detection methods of free fetal DNA and RNA in plasma and serum of pregnant women require high technical level of researchers, the detection method has a sensitivity of about 90%, and often requires the help of expensive high-throughput detection platforms or mass spectrometry detection platforms
Existing technologies are still unable to achieve efficient, accurate, fast and simple detection requirements

Method used

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  • Kit for non-invasive prenatal diagnosis of fetal trisomy in pregnant women and method of use thereof
  • Kit for non-invasive prenatal diagnosis of fetal trisomy in pregnant women and method of use thereof
  • Kit for non-invasive prenatal diagnosis of fetal trisomy in pregnant women and method of use thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0046] Example 1: Preparation of a kit for non-invasive prenatal diagnosis of fetal trisomy in pregnant women (for trisomy 21)

[0047] Primer pair PP 三体 with PP 常 The design, synthesis and screening steps are as follows:

[0048] (a) Taking human chromosome 21 and randomly selected chromosome 22 as objects, download the corresponding chromosome sequence information "NC_000021.9Homo sapiens chromosome 21,GRCh38.p2" and "NC_000022.11Homo sapiens chromosome 22, GRCh38.p2". The primer design software Primer Premier 6.0 was used to design primer pairs for chromosome 21 and chromosome 22 respectively for the downloaded DNA sequences. In order to ensure that the primers of the two chromosomes can have similar amplification efficiencies under the same conditions, the The design conditions are consistent: ①The melting point of the primer is 58±2°C, ②The length of the primer is 20-22nt, ③The length of the amplicon is 100bp. A total of 32 pairs of primers were designed and synthesiz...

Embodiment 2

[0059] Embodiment 2: Utilize the kit of embodiment 1 to carry out blood sampling to diagnose fetal trisomy 21 syndrome to pregnant women in the second trimester (18 weeks)

[0060] (1) Material

[0061] In this example, the primer pair combination of 19 / 46 was chosen.

[0062] Chromosome 21 primer pair 19: upstream primer 5'-GTCCCTTCACTGTCTGCCTACT-3' (SEQ ID No.1), downstream primer 5'-GCCAAGACTTGAGCCCATACTG-3' (SEQ ID No.2);

[0063] Chromosome 22 primer pair 46: upstream primer 5'-TCGGTGAGGCACATCAGCAT-3' (SEQ ID No.3), downstream primer 5'-GGACAGGACATGGTTGGTGAGA-3' (SEQ ID No.4);

[0064] Template samples: a. second-trimester serum (18 weeks of gestation) of a mother who gave birth to a healthy fetus, b. second-trimester serum (18 weeks of gestation) of trisomy 21 detected by amniocentesis.

[0065] (2) Template DNA extraction

[0066] Serum DNA of pregnant women was extracted using QIAamp DNA Kits (Qiagen, #51304), and the concentration of the obtained DNA sample was 15....

Embodiment 3

[0073] Embodiment 3: Utilize the kit of embodiment 1 to carry out blood collection diagnosis fetal trisomy 21 syndrome to pregnant women in the second trimester (16 weeks)

[0074] (1) Material

[0075] In this example, the primer pair combination of 19 / 46 was chosen.

[0076] Template samples: a. Serum from non-pregnant healthy women, b. Second-trimester serum (gestational week 16) with trisomy 21 detected by amniocentesis.

[0077] (2) Template DNA extraction

[0078] The serum DNA of the women was extracted using QIAamp DNA Kits (Qiagen, #51304), and the concentration of the obtained DNA sample was 11.9 ng / μL.

[0079] (3) Double fluorescence quantitative PCR

[0080] Reaction system 10μL, template DNA 1.0ng / μL, the above four primers respectively 0.2μM, 1×PCR mastermix; reaction conditions 95℃ for 3min, 95℃ for 15s→55℃ for 15s→72℃ for 45s for a total of 30 cycles, each sample was subjected to 10 Parallel test.

[0081] (4) High resolution melting curve analysis

[00...

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Abstract

The invention relates to a kit for noninvasive prenatal diagnosis of pregnant woman fetal trisomy syndrome. The kit comprises the following ingredients including an upstream and downstream primer pair PP trisomy designed by aiming one or several of No.21, No.18 or No.13 chromosomes, and an upstream and downstream primer pair PP autosome designed by aiming at any one autosome except the No.21, No.18 or No.13 chromosomes, wherein any one pair of PP trisomy and PP autosome needs to meet the following conditions that 1, the melting point difference values between every two primers of the four primers is within 4 DEG C; 2, the amplicon length is smaller than or equal to 150 bp and a non-target product is not generated; 3, the melting point difference value of the two amplicons is 2 DEG C or higher; 4, the melting peaks of the two amplicons are obviously separated, so the shoulder peak is not formed. The kit has the advantages that simplicity, convenience and high speed are realized; the accuracy and the sensitivity are high; the errors are small; the precision is high, so that the effective Down symdrome screening gestational week period is shortened by at least three weeks.

Description

technical field [0001] The invention belongs to a kit for prenatal diagnosis of fetal trisomy syndrome 21, 18 trisomy syndrome and 13 trisomy syndrome, in particular to a quantitative detection of the relative content of abnormal chromosomes and normal chromosomes in the blood of pregnant women with the possibility of trisomy kits and methods of use thereof. Background technique [0002] Chromosomal abnormal genetic disease is a kind of disease caused by abnormal number of chromosomes and morphological aberration, which can easily lead to spontaneous abortion, stillbirth and premature death, and is one of the important causes of congenital heart disease and mental retardation. With the development of biomedical technology, more and more chromosomal aberration diseases have been discovered. One of the more special is a class of autosomal trisomy diseases, mainly including trisomy 21, trisomy 18 and trisomy 13. Trisomy 21, also known as Down's Syndrome, is an incurable conge...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6883C12Q1/6851
CPCC12Q1/6851C12Q1/6883C12Q2537/143C12Q2531/113C12Q2563/107
Inventor 梁兴国王静范慧君
Owner 青岛千卓分子生物科技有限公司
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