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Primer group and kit for detecting osteogenesis insufficiency related genes based on multiple PCR (Polymerase Chain Reaction) and high-throughput sequencing technology and application of primer group and kit

A technology of osteogenesis imperfecta and a kit, which is applied in the direction of recombinant DNA technology, microbial measurement/inspection, biochemical equipment and methods, etc., can solve the problems of OI that need further epidemiological investigation, and achieve the prevention of osteogenesis imperfecta birth Defects, ensuring specificity and detection accuracy, and improving the effect of detection sensitivity

Pending Publication Date: 2022-07-08
苏州赛福医学检验有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The prevalence of OI in newborns is about 1 / 20 000 to 1 / 15000, but the prevalence of OI in my country still needs further epidemiological investigation

Method used

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  • Primer group and kit for detecting osteogenesis insufficiency related genes based on multiple PCR (Polymerase Chain Reaction) and high-throughput sequencing technology and application of primer group and kit
  • Primer group and kit for detecting osteogenesis insufficiency related genes based on multiple PCR (Polymerase Chain Reaction) and high-throughput sequencing technology and application of primer group and kit
  • Primer group and kit for detecting osteogenesis insufficiency related genes based on multiple PCR (Polymerase Chain Reaction) and high-throughput sequencing technology and application of primer group and kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Embodiment 1 Reagent kit and detection method

[0068] The invention provides a kit for detecting the pathogenic variants of osteogenesis imperfecta genes based on multiple PCR and high-throughput sequencing technology, and the detected osteogenesis imperfecta genes include IFITM5, WNT1, FKBP10, CRTAP, COL1A1 and COL1A2.

[0069] The kit includes specific primers as shown in Table 1.

[0070] In addition to the primers shown in Table 1, the kit provided by the present invention also includes enzymes and reagents commonly used in PCR reactions, such as universal primers, Index, dNTPs, high-fidelity DNA polymerase, Mg 2+ , PCR reaction buffer, etc.; may also contain standards and / or controls.

[0071] The specific primer pair information is shown in Table 1 below.

[0072] Table 1 Primer Information

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081]

[0082]

[0083]

[0084] When the sequences in Table 1 a...

Embodiment 2

[0110] 1. Obtain biological samples and nucleic acid extraction

[0111] For blood samples, saliva or other tissue samples obtained from hospitals or other research institutions, the genomic DNA in each blood sample was extracted by magnetic bead method, and the concentration and purity of DNA were measured by spectrophotometer, and the OD of genomic DNA of each sample was obtained. 260 / OD 280 All are located between 1.7-2.0, the concentration is not less than 20ng / microliter, and the total amount is not less than 1 microgram.

[0112] 2. Target gene enrichment PCR:

[0113] Since the areas amplified by the 158 pairs of primers are too close or overlap each other, the primer pairs are divided into three groups, and Pool-1, Pool-2, and Pool-3 are used as primers for PCR amplification. The PCR reaction system performs the first step of PCR amplification reagent configuration. First, add the required reagents into a new centrifuge tube. After the reagent configuration is compl...

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Abstract

The invention relates to the field of biological detection, in particular to a primer group and a kit for detecting osteogenesis insufficiency related genes based on multiple PCR (polymerase chain reaction) and high-throughput sequencing technologies and application of the primer group and the kit. According to the multiplex PCR specific primer for detecting the osteogenesis imperfect gene based on the high-throughput sequencing technology, the detection gene range comprises detection of all exons of IFITM5, WNT1, FKBP10, CRTAP, COL1A1 and COL1A2 genes; a plurality of samples and a plurality of osteogenesis imperfect susceptibility genes can be detected at the same time, the detection efficiency and accuracy are effectively improved, meanwhile, the cost is reduced, and the operation steps are simplified.

Description

technical field [0001] The invention relates to the field of biological detection, in particular to a primer set, a kit and an application for detecting osteogenesis imperfecta-related genes based on multiplex PCR and high-throughput sequencing technology. Background technique [0002] Osteogenesis imperfecta (OI), also known as brittle bone disease, is the most common single-gene inherited bone disease. It is caused by a variety of pathogenic gene mutations that lead to a decrease in the quantity or quality of bone matrix proteins, resulting in low bone mass. , bone disease characterized by increased bone fragility and repeated fractures. Most are autosomal dominant, a few are autosomal recessive, and rare are X chromosome sex-linked. [0003] Bones are mainly composed of organic and inorganic matter. Type I collagen (COL1) is the most abundant component in bone organic matter, accounting for more than 90% of bone organic matter, and is essential for maintaining the integ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/686C12Q1/6869C12N15/11
CPCC12Q1/6883C12Q1/686C12Q1/6869C12Q2600/16C12Q2600/156C12Q2537/143C12Q2563/143C12Q2563/149C12Q2535/122
Inventor 杨锋余伟师梁萌萌
Owner 苏州赛福医学检验有限公司
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