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Set of primer group and method capable of simultaneously detecting mediterranean anemia, epicophosis, phenylketonuria and hepatolenticular degeneration

A technology for phenylketonuria and hepatoloids, which is applied in biochemical equipment and methods, recombinant DNA technology, and microbial measurement/inspection, can solve problems such as poor prognosis, and achieve good repeatability and high efficiency.

Active Publication Date: 2018-04-24
GUANGZHOU DARUI BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The current treatment drugs are mainly copper ion chelating agent penicillamine and ion exchanger zinc sulfate. The prognosis is closely related to the diagnosis and treatment. If early diagnosis and treatment can be started, especially in the early stage of symptoms, most of them will have a good prognosis; If the treatment is late, important organs such as liver, brain and kidney have been irreversibly damaged, and at most only partial improvement of symptoms can be obtained, and the prognosis is poor

Method used

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  • Set of primer group and method capable of simultaneously detecting mediterranean anemia, epicophosis, phenylketonuria and hepatolenticular degeneration
  • Set of primer group and method capable of simultaneously detecting mediterranean anemia, epicophosis, phenylketonuria and hepatolenticular degeneration
  • Set of primer group and method capable of simultaneously detecting mediterranean anemia, epicophosis, phenylketonuria and hepatolenticular degeneration

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] The design of embodiment 1 primer set

[0046] 1. Determine the detection range of genes related to the four genetic diseases of thalassemia, deafness, phenylketonuria and hepatolenticular degeneration; design primers for each mutation site of the relevant pathogenic genes of these genetic diseases , to avoid the formation of primer-dimers and primers with high GC content, and finally select primers with an annealing temperature of 60 °C. All designed primers were compared by BLAST on the NCBI database. There should be no complementary sequences between the primers and they should have high specificity. The primer pairs adjacent to the position of the human genome Hg19 reference sequence were assigned to different primer pools to avoid the generation of overlap, and the primer pairs with similar GC content were assigned to the same primer pool. Finally, the primer set of the present invention is optimized, including three primer pools: primer pool I, primer pool II and...

Embodiment 2

[0063] Example 2 Detection method for genes related to thalassemia, deafness, phenylketonuria, and hepatolenticular degeneration

[0064] Using the primer set designed in Example 1, the peripheral whole blood genomic DNA of the sample was used as a template, combined with the multiplex PCR method to construct the library, and the high-throughput sequencing method was used to sequence the library, and the sequencing data was analyzed to detect and analyze gene mutations.

[0065] 1. Reagents used in this method: DNeasy Blood&Tissue Kit, Qubit dsDNA HS AssayKit, Ion Ampliseq Library Kit 2.0, Ion Xpress Barcode adapters1-16, absolute ethanol, AMPure XP beads, Ion PI HI-Q Sequencing 200Kit, Ion PI HI -Q OT2 200Kit, Dynabeads Myone Streptavidin C1, Ion PI Chip Kit v2, Isopropanol, Sodium Hydroxide, etc.

[0066] 2. Detection method

[0067] (1) The detection template is DNA from peripheral whole blood;

[0068] (2) Amplification of the target sequence

[0069] For each sample, t...

Embodiment 3

[0111] Example 3 Application of detection method in wild-type human genome

[0112] 1. Collection, transportation and preservation of specimens:

[0113] (1) Specimen collection: The specimen is human peripheral whole blood, and the above 58 loci were tested by clinical first-generation sequencing, and the results were all wild type. 2 mL of venous blood was taken routinely and treated with EDTA anticoagulant.

[0114] (2) Storage: It can be detected immediately, and stored at -20±5°C for 2 years.

[0115] (3) Transportation: Store and transport at 4°C.

[0116] 2. Extraction of human genomic DNA

[0117] DNA was extracted using DNeasy Blood & Tissue Kit extraction kit. The specific steps are as follows: Add 20 μL proteinase K, 200 μL whole blood sample and 20 μL AL buffer to a 1.5mL EP tube, mix well and incubate at 56°C for 10 minutes; add 200 μL absolute ethanol, mix well and transfer all the liquid to the spin column , centrifuge at 8000rpm for 1 minute, discard the l...

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Abstract

The invention discloses a set of primer group and a detection method capable of simultaneously detecting relevant gene mutation of mediterranean anemia, epicophosis, phenylketonuria and hepatolenticular degeneration. The primer group is composed of a primer pool 1 comprising 9 pairs of primers, a primer pool 2 comprising 10 pairs of primers, and a primer pool 3 comprising 8 pairs of primers, wherein upstream and downstream primer sequences are sequentially shown as SEQ ID NO.1-54. The three primer pools of the primer group can carry out multiplex PCR amplification simultaneously, in combination with a high-throughput sequencing technology, common pathopoiesis mutation sites capable of simultaneously detecting the four genetic diseases including mediterranean anemia, epicophosis, phenylketonuria and hepatolenticular degeneration are established, and in addition, the method is efficient, rapid and accurate, is good in repeatability, and has a very good application prospect.

Description

technical field [0001] The invention belongs to the technical field of disease detection. More specifically, it relates to a set of primers and a detection method for simultaneously detecting genes related to thalassemia, deafness, phenylketonuria, and hepatolenticular degeneration. Background technique [0002] Thalassemia (referred to as "thalassemia") is the most common single-gene genetic disease in the world and has the greatest impact on human health. It is also a genetic disease recommended by the World Health Organization (WHO) for priority prevention. Clinically, α-thalassemia and β-thalassemia are the most common, and the related genes are HBA2 and HBB, respectively. Deafness is a common disease that affects human health and disability, and is also the most common genetic disease. According to statistics, the incidence of deafness in newborns is 1‰, and the genes that affect it mainly include GJB2, GJB3 and SLC26A4, etc.; deafness gene diagnosis The results have ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/686C12Q1/6869C12Q1/6883C12Q2600/156C12Q2537/143C12Q2535/122
Inventor 李明吴英松杨学习杨旭叶倩平
Owner GUANGZHOU DARUI BIOTECH
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