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Primer, kit and detection method for detecting IDH1 gene variation through ddPCR technique

A technology of technical detection and genetic variation, applied in the fields of medicine and biology, can solve the problems of high labor intensity, unsuitability for high-throughput detection and large-scale clinical application, and achieve the effects of reducing pain, good specificity, and simple operation

Inactive Publication Date: 2018-07-31
PRIMBIO GENES BIOTECH WUHAN CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the labor intensity is slightly higher, professional technicians are required, and it is not suitable for high-throughput detection and large-scale clinical application

Method used

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  • Primer, kit and detection method for detecting IDH1 gene variation through ddPCR technique
  • Primer, kit and detection method for detecting IDH1 gene variation through ddPCR technique
  • Primer, kit and detection method for detecting IDH1 gene variation through ddPCR technique

Examples

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Embodiment 1

[0048] Example 1: Design of primers and probes

[0049] In this example, primers suitable for the quantitative detection of IDH1 gene variation on the ddPCR technology platform were designed based on the wild-type gene sequence of exon No. 4 of the IDH1 gene and the corresponding mutant gene sequence; the Taqman probe method was used to design primers for IDH1 mutants Design mutant and wild-type probes corresponding to the wild-type sequence.

[0050] The upstream and downstream primers for amplifying the IDH1 R132H mutant gene and amplifying the wild gene:

[0051] Upstream primer SEQ NO1: 5'-ttgccaacatgacttacttgatcc-3'

[0052] Downstream primer SEQ NO2: 5'-aaatatcccccggcttgtgag-3'

[0053] The IDH1 mutant gene detection probe includes FAM fluorescent group, mutation site binding sequence, and MGB quenching group in sequence from the 5' end to the 3' end. This probe binds to the gene mutant site during annealing;

[0054] SEQ NO3: 5'-FAM-ataagcatgacgacctatgat-MGB-3'

[0...

Embodiment 2

[0058] Example 2: Detection of IDH1 R132H Gene Variation in Whole Blood Samples

[0059] 1. Prepare the sample to be tested: the DNA containing the internal reference human IDH1 gene, the DNA of the human IDH1 R132H mutant gene, and the DNA mixed sample containing the internal reference human IDH1 gene and the human IDH1 R132H mutant gene (wherein the DNA of the human IDH1 R132H mutant gene is mixed with the DNA containing the human IDH1 R132H mutant gene The content ratios of the internal reference human IDH1 gene were 1 / 100 and 1 / 1000, respectively). The DNA is derived from human peripheral blood, wherein the DNA template of the human IDH1 R132H mutant gene is derived from an IDH1 R132H gene mutant cell line (identified by PCR sequencing).

[0060] 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.

[0061] 3. Configure the PCR master mix

[0062] 20 μL of PC...

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Abstract

The invention relates to the technical field of medicines and biology, in particular to a primer, kit and detection method for detecting an IDH1 gene variation through the ddPCR technique. The primerand a probe, provided by the invention, for detecting the IDH1 gene variation through the ddPCR technique is designed according to the sequence of a wild type gene and the sequence of a correspondingmutant gene of a fourth exon of the IDH1 gene, the various mutations comprising the fourth exon like R132H, R132C, R132S, R132G, R132L and R132V of the fourth exon of the IDH1 gene are detected in combination with a PCR platform, and the mutant DNA of the IDH1 gene carried in a sample is absolutely quantified; an amplified fragment of the primer is 91bp and is particularly suitable for the amplification of the small-fragment DNA sample like the plasma free DNA, the quite low abundance IDH1 gene mutation is detected from the cfDNA, and the primer has the characteristics of good specificity andhigh sensitivity, can be applied to the absolute quantitative detection of the IDH1 gene mutation on a ddPCR platform, and plays very important roles in directing clinical treatment and improving theprognosis of a patient.

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 multiple gene variations of IDH1 by ddPCR technology. Background technique [0002] 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 IDH substrates to participate in different biological pathways and exert different biological functions. In mammalian cells, IDH isozymes have the following three forms: NAD-dependent mitochondrial IDH1, NADP-dependent mitochondrial IDH2, and NADP-dependent cytoplasmic IDH3. The gene encoding NADP-dependent human isocitrate dehydrogenase 1 (IDH1) is located on chromosome 2q33.3. The IDH1 gene mutations discovered so far all occur at the R132 position of exon 4, an...

Claims

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Application Information

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IPC IPC(8): C12Q1/6858C12Q1/6851C12N15/11
CPCC12Q1/6851C12Q1/6858C12Q2545/101C12Q2563/159C12Q2563/107
Inventor 王昕昀
Owner PRIMBIO GENES BIOTECH WUHAN CO LTD