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Primer combination for embryo preimplantation hereditary hearing loss gene detection, kit and application

A technology for hereditary deafness and preimplantation, applied in the field of molecular biology, can solve the problems of high risk of misdiagnosis, time-consuming, complicated operation, etc., so as to reduce the interference of results, improve the accuracy, and reduce the risk of misdiagnosis.

Active Publication Date: 2018-12-21
SUZHOU BASECARE MEDICAL DEVICE CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Hereditary deafness is the main birth defect in my country, and the prognosis and treatment are difficult to achieve complete recovery. Therefore, tertiary prevention of deafness and birth defects has been carried out in China
However, traditional PGD detection mainly uses PCR-based next-generation sequencing technology or capillary electrophoresis to detect STR to complete the analysis. The detection throughput is low, the operation is complicated, and it takes a long time. It is usually impossible to realize the simultaneous detection and detection of pathogenic variants and polymorphic sites. analyze
This method can only detect a small number of loci, so the risk of misdiagnosis caused by allele dropout is relatively high

Method used

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  • Primer combination for embryo preimplantation hereditary hearing loss gene detection, kit and application
  • Primer combination for embryo preimplantation hereditary hearing loss gene detection, kit and application
  • Primer combination for embryo preimplantation hereditary hearing loss gene detection, kit and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0059] Example 1 Analysis of pathogenic variants in family samples

[0060] 1. Sample preparation

[0061] The blood genomic DNA of both husband and wife and their affected offspring (proband) was extracted, the DNA concentration was measured by Qubit 2.0, and a certain amount was taken as the DNA sample to be tested.

[0062] 2. Target sequence capture and library construction

[0063] 1. Target Enrichment Capture

[0064] 1) After the DNA sample was vortexed and centrifuged, place it on an ice box, take two 0.2ml PCR tubes for each sample, and prepare the reaction system according to Table 3:

[0065] table 3

[0066] components

Reaction volume (μl)

dna sample

2(50ng)

nuclease free water

4

5× Amplification Mixture

4

[0067] 2) After the preparation is completed, evenly distribute to two 0.2ml PCR tubes, 5μl in each tube. Add 5 μl of primer pool 1 to one tube, and 5 μl of primer pool 2 into the other tube, vortex to mix, ...

Embodiment 2

[0101] Example 2 Pre-implantation detection of embryos

[0102] 1. Sample Preparation

[0103] Three to five cells from the trophectoderm of blastocyst-stage embryos were isolated as samples to be tested. The blastocyst stage embryos were sampled according to conventional embryo biopsy techniques, and the cells taken out were washed with 1×PBS (without Ca 2+ , Mg 2+ ) and washed 3 times, placed in a 0.2ml PCR tube with a volume not exceeding 2.5μl. Single-cell samples first use PicoPlex TM WGA kit for whole genome amplification.

[0104] 2. Target sequence capture and library construction

[0105] With embodiment 1.

[0106] The joints used for the 8 embryo samples to be tested G30218032601P01 to G30218032601P08 are SEQ ID NO.11 to SEQ ID NO.18 respectively.

[0107] 3. Fluorescent quantitative PCR to measure library concentration

[0108] With embodiment 1.

[0109] 4. On-machine sequencing and analysis of sequencing results

[0110] Dilute the library to 100pM, ta...

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Abstract

The invention relates to a primer combination for embryo preimplantation hereditary hearing loss gene detection, a kit and application. A primer pair for PCR amplification is designed aiming at the coding region sequence of the hearing loss virulence genes GJB2 and SLC26A4, and the related SNP site on the 7th and / or 13th chromosome, enrichment can be conducted on the virulence gene target region through a target capturing technique, a library is built, then embarkation sequencing and haplotype analysis are conducted, and the primer combination can further be used for analyzing the related geneinformation, and has the important guidance and research value on auxiliary diagnosis and treatment of hereditary hearing loss. The primer combination can achieve synchronous detection and analysis of pathogenic variants and polymorphic sites, so that detection and linkage analysis of the pathogenic variants can be complete in the same reaction system, interference of artificial factors to the result can be reduced, the detection accuracy of detection can be improved, and the misdiagnose risks caused by ADO can be effectively decreased.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to a primer composition, kit and application for preimplantation hereditary deafness gene detection. Background technique [0002] Hereditary deafness is a large class of genetic diseases. According to WHO statistics, in 2013, the number of hearing disabled people in the world reached 360 million, accounting for 5.3% of the world's total population. Worldwide, 1 out of every 1,000 newborns is born with congenital deafness. children. According to statistics from various countries, congenital hearing impairment ranks first among birth defects, with an incidence rate of about 1‰~3‰. According to the results of the second national sample survey of disabled people released in December 2006, there are 27.8 million people with hearing and speech disabilities in my country, accounting for 33.52% of the total number of disabled people, and 900,000 new cases of birth defects each year, inclu...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/156
Inventor 曹扬冒燕刘慧敏孔令印梁波
Owner SUZHOU BASECARE MEDICAL DEVICE CO LTD
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