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Kit for detecting achondroplasia (ACH) and hypochondroplasia (HCH) of neonates

A reagent and fetal technology, applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc., to achieve high detection sensitivity, high accuracy, and good specificity

Inactive Publication Date: 2017-11-03
BIOYONG TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although the accuracy of MALDI‐TOF is as high as 0.1% to 0.01%, which is much higher than that of SDS electrophoresis and high performance gel chromatography, which are routinely used at present, this mass spectrometry technology is mainly aimed at protein or polypeptide diseases, and there is no use of this technology at present. Report on the Diagnosis of FGFR3 Mutation Site by Mass Spectrometry

Method used

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  • Kit for detecting achondroplasia (ACH) and hypochondroplasia (HCH) of neonates
  • Kit for detecting achondroplasia (ACH) and hypochondroplasia (HCH) of neonates
  • Kit for detecting achondroplasia (ACH) and hypochondroplasia (HCH) of neonates

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0080] Example 1: Primer design and synthesis.

[0081] For the four mutation types at bases 1108, 1138 and 1620 of the FGFR3 gene, the corresponding specific PCR primer core sequences (SEQ ID No: 1 to SEQ ID No: 6) and specific extension primer core sequences (SEQ ID No: 7 to SEQ ID No: 9).

[0082] Among them, in order to prevent the PCR primers from entering the detection window of the mass spectrometer and interfering with the detection effect, a certain number of bases can be added to the core sequence (SEQ ID No: 1 to SEQ ID No: 6) at the 5' end of each PCR primer , common such as 10bp tag (ACGTTGGATG), to increase the molecular weight of the PCR primer, thereby exceeding the detection window of the mass spectrometer.

[0083] Related primers were synthesized at Sangon Bioengineering (Shanghai) Co., Ltd.

[0084] The following detection process is performed with reference to the "Folic Acid Genetic Metabolism Gene Mutation Detection Kit (Time-of-Flight Mass Spectrometr...

Embodiment 2

[0085] Example 2: Extraction of Free DNA from Peripheral Plasma of Pregnant Women

[0086] The blood sheet used is a circular filter paper with a diameter of 2 cm. Take 50ul of whole blood, drop it on the center of the circular filter paper, and then dry it for later use. The specific steps of blood sheet DNA extraction are as follows:

[0087] (1) Prepare the scissors and pre-treat them with 75% alcohol. Then cut out the circular piece of paper with blood spots and cut it into small pieces, put it into a 1.5ml centrifuge tube, add 20ul proteinase K, 20ulDTT and 500ul Lysis Buffer 1, mix well (vortex) , placed at 56°C for 2h (during which the mixture was inverted and mixed 3-4 times).

[0088] (2) Take out the centrifuge tube, centrifuge briefly, and use a pipette to transfer the supernatant as completely as possible to a new 1.5ml centrifuge tube to remove the interference of the filter paper on the next step of the experiment.

[0089] (3) Add 850ul Binding Buffer 2 and 1...

Embodiment 3

[0098] Embodiment three: Biological experiment.

[0099] Using ABI 9700 PCR instrument, according to the instruction manual, the 3 target detection sites of FGFR3 gene were tested.

[0100] The components used in the kit for PCR, PCR product purification and single base extension are shown in Table 5:

[0101] table 5

[0102]

[0103] According to the manual, the specific operation method is as follows:

[0104] 1. PCR amplification

[0105] 1.1 In the PCR preparation area, prepare 200ul PCR reaction tubes according to the number of samples to be tested (including positive quality control products, negative controls, and blank controls), and mark the sample number on the tube;

[0106] 1.2 Take out the PCR mixture and PCR enzyme from the kit, let it thaw naturally, vortex and oscillate to mix well, and centrifuge instantly to the bottom of the tube;

[0107] 1.3 According to the number of samples, take out the PCR primer mixture and PCR reaction solution according to t...

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Abstract

The invention discloses a primer system for detecting gene mutation sites related to neonatal achondroplasia (ACH) and hypochondrosis (Hypochondroplasia, HCH). Based on the products prepared by this primer system, by using time-of-flight mass spectrometry (MALDI‐TOF‐MS) technology, the 1108th base G>T and the 1138th base of the fetal FGFR3 gene in the peripheral plasma free DNA of pregnant women can be detected G>A and G>C and the 1620th base C>A, a total of 4 mutation types are detected. This method has the characteristics of high sensitivity, high accuracy, high resolution, concise map, wide mass range and fast speed. . The simple, microquantizable, large-scale, parallelized and highly automated processing of biological samples to be tested is of great significance for the non-invasive prenatal diagnosis of ACH / HCH.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a detection method and product for detecting gene mutations related to neonatal achondroplasia (ACH) and hypochondroplasia (HCH). Specifically, the integrated multiplex PCR technology, single base extension technology and mass spectrometry technology are used to detect the 1108th base G>T, the 1138th base G>A and the fetal FGFR3 gene in the cell-free DNA of the peripheral plasma of pregnant women. G>C and the 1620th base C>A, a total of 4 types of mutation methods and corresponding kits. Background technique [0002] my country is a country with a high incidence of birth defects. It is estimated that the current incidence of birth defects in my country is about 5.6%, and the number of new birth defects is about 900,000 each year. Among them, single-gene genetic diseases are the most important type of birth defects. According to WHO statistics, The cumulative incidence of mo...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6883C12Q2600/156
Inventor 马庆伟钟逾吴娜拉胡李座详张海燕谭甜霞
Owner BIOYONG TECH
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