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Primers, reagent kit and method for detecting IDH1 (isocitrate dehydrogenase 1) R132H gene variation by aid of ddPCR (droplet digital polymerase chain reaction) technologies

A technology for technical detection and gene variation, applied in the medical and biological fields, can solve the problems of difficult to detect specific types and sequences of cell-free DNA, blood-derived DNA interference, etc., and achieve the effect of guiding clinical treatment, reducing pain, and simple operation.

Inactive Publication Date: 2018-08-10
PRIMBIO GENES BIOTECH WUHAN CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the extremely low content of plasma cell-free DNA in the blood and the interference of a large amount of blood-derived DNA, it is still difficult to separate and purify DNA with high-efficiency DNA enrichment methods and high-sensitivity detection methods (such as sequencing, ARMS-PCR). Accurate and reliable detection of specific species and sequences of tumor-associated cell-free DNA

Method used

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  • Primers, reagent kit and method for detecting IDH1 (isocitrate dehydrogenase 1) R132H gene variation by aid of ddPCR (droplet digital polymerase chain reaction) technologies
  • Primers, reagent kit and method for detecting IDH1 (isocitrate dehydrogenase 1) R132H gene variation by aid of ddPCR (droplet digital polymerase chain reaction) technologies
  • Primers, reagent kit and method for detecting IDH1 (isocitrate dehydrogenase 1) R132H gene variation by aid of ddPCR (droplet digital polymerase chain reaction) technologies

Examples

Experimental program
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Effect test

Embodiment 1

[0043] In this example, primers suitable for the quantitative detection of R132H gene variation on the ddPCR technology platform were designed for the upstream and downstream of the target region of the human IDH1 R132H gene; the primers were designed using the Taqman probe method, and the corresponding primers were designed for the IDH1 R132H mutant and wild-type sequences Mutant and wild-type probes.

[0044] The upstream and downstream primers for amplifying the IDH1 R132H mutant gene and amplifying the wild gene:

[0045] Upstream primer SEQ NO1: 5'-ccaacatgacttacttgatcccc-3'

[0046] Downstream primer SEQ NO2: 5'-aatatcccccggcttgtgag-3'

[0047] The IDH1 R132H mutation gene detection probe includes FAM fluorescent group, mutation site binding sequence, and MGB quenching group from the 5' end to the 3' end. This probe binds to the gene mutation site during annealing ;

[0048] SEQ NO3: 5'-FAM-taagcatgatgacctatgat-MGB-3'

[0049] The wild-type detection probe includes a...

Embodiment 2

[0052] Example 2: Detection of IDH1 R132H Gene Variation in Whole Blood Samples

[0053] 1. Prepare the samples to be tested: DNA containing wild-type human IDH1 R132H gene, DNA of human IDH1 R132H mutant gene, and wild-type mutant DNA mixed samples (wherein the content ratio of mutant and wild type is 1 / 100, 1 / 100, respectively. 1000, 1 / 10000). The DNA is derived from human peripheral blood, wherein the DNA template of the human IDH1R132H mutant gene is derived from an IDH1 R132H gene mutant cell line (identified by PCR sequencing).

[0054] 2. Extraction of cfDNA: cfDNA was extracted using a kit, and cfDNA was extracted referring to the instructions of the QIAamp Circulating Nuleacid Kit kit from QIAGEN.

[0055] 3. Configure the PCR master mix

[0056] 20 μL of PCR master mix includes: 10 μL of 2× digital PCR master mix (Biorad, #1863010), 500 nM upstream primers for amplifying IDH1R132H mutant gene and wild gene, downstream primers for amplifying IDH1 R132H mutant gene a...

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Abstract

The invention discloses primers, a reagent kit and a method for detecting IDH1 (isocitrate dehydrogenase 1) R132H gene variation by the aid of ddPCR (droplet digital polymerase chain reaction) technologies. The method includes extracting cfDNA (cell-free deoxyribonucleic acid) in peripheral blood; designing two probes and a pair of primers for IDH1R132H mutant types and wild types; carrying out ddPCR amplification by the aid of the cfDNA used as a template; carrying out data analysis on obtained amplification products to obtain absolute quantities and proportions of IDH1R132H mutant genes. Thereagent kit for detecting the IDH1R132H gene variation is based on the method. The primers, the reagent kit and the method have the advantages that provided primer amplification fragments 83bp are particularly applicable to amplifying small-fragment DNA samples such as plasma free DNA and can be combined with the detection probes, accordingly, the IDH1R132H gene variation with extremely low abundance can be detected from the cfDNA, the primers, the reagent kit and the method are good in specificity and high in sensitivity, human IDH1R132H gene variation can be absolutely quantitatively detected by the aid of the primers, the reagent kit and the method, and important effects can be realized by the primers, the reagent kit and the method for guiding clinical treatment and improving patientprognosis; the IDH1R132H gene variation can be detected only by the aid of the peripheral blood without tissue specimen collection, and accordingly the method is particularly suitable for patients intolerant to tissue sampling.

Description

technical field [0001] The invention relates to the fields of medicine and biotechnology, in particular to a primer, a kit and a detection method for detecting IDH1 R132H gene variation by ddPCR technology. Background technique [0002] Glioma is the most common primary central nervous system tumor, accounting for about 35% to 60% of all primary intracranial tumors, with an adult incidence of 8 to 10 / 100,000 and a 5-year survival rate of 20% to 30%, and its incidence rate is still on the rise in recent years. The World Health Organization (WHO) has established clinical guidelines for classifying gliomas into various subtypes and grading them from I to IV indicating their degree of malignancy. [0003] Isocitrate dehydrogenase (IDH) is an enzyme family that plays an important role in the tricarboxylic acid cycle, not only plays an important role in energy metabolism, amino acid and vitamin synthesis, but also regulates the activity of the enzyme will Directly affect IDH or ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6858C12N15/11
CPCC12Q1/6858C12Q1/6886C12Q2600/118C12Q2600/156C12Q2531/113C12Q2563/159C12Q2563/107
Inventor 王昕昀
Owner PRIMBIO GENES BIOTECH WUHAN CO LTD
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