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Kit, primers, probe sequence and method for detecting fetus chromosome aneuploid

A technology for aneuploidy and chromosomes, which is applied in the field of chromosome detection, can solve the problems of high investment cost, inaccurate detection, and high requirements for high-throughput screening technology operations, so as to achieve short time consumption, avoid detection errors, and reduce manpower input Effect

Active Publication Date: 2016-06-22
内蒙古因诺生物技术有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] The purpose of the present invention is to overcome the problem of inaccurate detection in serum screening technology and the shortcomings of high-throughput screening technology with high operational requirements and huge investment costs, and to provide a simple, low-cost, fast and high-accuracy reagent Box, primer and method of use thereof, used for digital PCR platform to detect fetal chromosome number variation

Method used

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  • Kit, primers, probe sequence and method for detecting fetus chromosome aneuploid
  • Kit, primers, probe sequence and method for detecting fetus chromosome aneuploid
  • Kit, primers, probe sequence and method for detecting fetus chromosome aneuploid

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Effect test

Embodiment 1

[0045] Embodiment 1 detects the design of primer and probe

[0046] On the one hand, the present invention designs and provides a kit for detecting ploidy of fetal chromosome number, and designs amplification primers and taqman probes on chromosomes 13, 18 and 21. In the plasma, the free DNA of the mother and the fetus is not a complete chromosome and a large fragment, so multiple detection sites can be designed on one chromosome, so that the number of chromosomes 13, 18 and 21 can be detected more accurately. In the present invention, 10 qualified amplification primers and probes were designed and screened on chromosomes 13, 18 and 21 respectively.

[0047] In order to screen out detection reactions with approximately the same amplification efficiency, the present invention designs 20 detection primers and probes on one chromosome, each detection is tested separately, and the amplification efficiency is high and the amplification efficiency is similar. Primer and probe detec...

Embodiment 2

[0140] Example 2 Peripheral blood of pregnant women is used for plasma DNA extraction

[0141] a. Take 10ml of peripheral blood from pregnant women at 12-23 weeks of pregnancy into EDTA anticoagulant tubes, mix upside down 8 times, and store at room temperature; b. Separate peripheral blood plasma, centrifuge at 1600g for 10min at room temperature, and centrifuge the supernatant (plasma ) into 1.5ml centrifuge tubes, centrifuge again at 16000g at room temperature for 10min to remove residual cells, collect the supernatant obtained by centrifugation and divide into 1.5ml centrifuge tubes, the volume of plasma in each tube is 450μL; c, extract DNA in the obtained plasma was collected and quantified. The extraction of plasma DNA adopts the adsorption column method, and the specific steps are to melt 450ul of serum samples stored in a refrigerator at room temperature. Centrifuge at 16000g for 5min at room temperature, transfer 400-430ul to a new 1.5ml centrifuge tube. Add 40μl o...

Embodiment 3

[0142] Embodiment 3 Preparation of reaction system test template

[0143] Using Tiangen Blood Genomic DNA Extraction Kit (DP318-02) to extract the whole genome DNA as the test template of the reaction system, use nanodrop to measure the DNA, the OD range of DNA is 1.7-2.0, dilute the final concentration to 25ng / μl, and set aside.

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Abstract

The invention provides a kit, primers, probe sequences and a method for detecting fetus chromosome aneuploid. The kit comprises the multiple pairs of specific primers and a plurality of specific Taqman probes all of which are designed according to 13#, 18# and 21# chromogene sequences of the human, and further comprises a reaction system used for digital PCR, wherein the nucleotide sequence of the specific primers and the specific Taqman probes is SEQ ID NO: 1-90. The detection method comprises the steps that firstly, the specific primers and the specific Taqman probes are designed according to the 13#, 18# and 21# chromogene sequences of the human; secondly, peripheral blood of a (12-23)-week pregnant woman is collected for plasma DNA extracting; thirdly, the reaction system of the digital PCR amplification gene sequence is prepared; fourthly, the numbers of 13#, 18# and 21# chromosomes of a fetus are calculated through digital PCR detection for the number of the chromosomes, digital PCR is used for detecting FAM and VIC fluorescence of all reaction units of a chip, and the copy number of the 13# chromosome, the copy number of the 18# chromosome and the copy number of the 21# chromosome are calculated respectively. By means of the kit and the method, the number of the 13# chromosome, the number of the 18# chromosome and the number of the 21# chromosome of the fetus can be efficiently, quickly and accurately detected without invasion, and the kit and the method are beneficial to clinic application and popularization.

Description

technical field [0001] The invention relates to chromosome detection, in particular to the detection of fetal free nucleic acid in maternal blood, especially the method for detecting fetal aneuploidy by digital PCR technology. Background technique [0002] Birth defects are an urgent problem to be solved in the current prenatal and postnatal care, and chromosomal abnormalities are an important cause of birth defects. According to statistics, the incidence of chromosomal abnormalities in newborns is about 0.5. Among the many chromosomal abnormalities, disability and life obstacles caused by chromosomal aneuploidy are particularly obvious. Among them, trisomy 21 (Down syndrome), trisomy 18 (Edward's syndrome) and trisomy 13 (Patau syndrome) are the most common autosomal aneuploidies. The incidence rates in China are 1 / 800, 1 / 6000 and 1 / 10000 respectively. Although chromosomal aneuploidy occurs at all maternal ages, the incidence of chromosomal aneuploidy increases significa...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6851C12Q2531/113C12Q2537/16C12Q2561/101C12Q2563/159
Inventor 刘涛
Owner 内蒙古因诺生物技术有限公司
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