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Primer probe for detecting amplification of recurrence and metastasis gene FZD2 of neuroblastoma, and application of primer probe

A technology for neuroblastoma, recurrence and metastasis, applied in recombinant DNA technology, microbial assay/examination, biochemical equipment and methods, etc., can solve problems such as developmental defects and cancer, and achieve the effect of avoiding PCR false negatives

Pending Publication Date: 2021-08-24
SHENGZHEN CHINA GENE TECH COMPANY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Mutations in components of the WNT pathway lead to specific developmental defects, while aberrant WNT signaling often leads to cancer

Method used

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  • Primer probe for detecting amplification of recurrence and metastasis gene FZD2 of neuroblastoma, and application of primer probe
  • Primer probe for detecting amplification of recurrence and metastasis gene FZD2 of neuroblastoma, and application of primer probe
  • Primer probe for detecting amplification of recurrence and metastasis gene FZD2 of neuroblastoma, and application of primer probe

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Example 1 Design of specific primers and probes for detecting the amplification of neuroblastoma recurrence and metastasis gene FZD2

[0034] With the human FZD2 as the target gene and the human EFTUD2 gene as the internal reference gene, specific primers and probes suitable for ddPCR were designed. The sequences of the primers and probes are as follows:

[0035]Target gene upstream primer sequence FZD2-F: 5'-TTTCTGTCGGGCTGCTACAC-3'SEQ ID NO: 1,

[0036] Target gene downstream primer sequence FZD2-R: 5'-GTAACCGTCCTCGGAGAAGC-3'SEQ ID NO: 2,

[0037] Target gene probe sequence FZD2-P: 5'-FAM-GCTCCAGGAGCGCGTGGTGTGCA-BHQ1-3'SEQ ID NO: 3, internal reference gene upstream primer sequence EFTUD2-F: 5'-CTCAAAGTGCGGGGACTGAT-3'SEQ ID NO: 4,

[0038] Internal reference gene downstream primer sequence EFTUD2-R: 5'-GGCATCAGGGTGACTCCAAA-3'SEQ ID NO: 5,

[0039] Internal reference gene probe sequence EFTUD2-P: 5'-HEX-AGCCTGCTTCCTGGGAATGTGCTGCT-BHQ1-3'SEQ ID NO:6.

Embodiment 2

[0040] Example 2 Detection of the Amplification of Neuroblastoma Recurrent and Metastatic Gene FZD2 Using Digital PCR Detection Primers and Probes

[0041] 1. DNA extraction from plasma

[0042] The NucleoSpin Plasma XS kit (product number: 740901.50) produced by MNG Company was used to extract nucleic acid from plasma samples. Take 240 μl of plasma, perform nucleic acid extraction according to the instructions of the extraction kit, and finally resuspend with 20 μl of eluent.

[0043] 2. Digital PCR amplification reaction

[0044] (1) Prepare digital PCR reaction solution; 20 μl of digital PCR reaction solution is formulated as follows: 4 μl of one-step reaction buffer, 2 μl of enzyme mixture, 1.2 μl of 10 μM FZD2-F, FZD2-R, EFTUD2-F and EFTUD2-R primers each, 0.6 μl each of 10 μM FZD2-P and EFTUD2-P, 8 μl of the DNA template of the sample to be tested;

[0045] (2) On the generation chip of droplet PCR, add 20 μ L of droplet PCR reaction system in step 1), then add 40 μ L...

Embodiment 3

[0051] Example 3 Neuroblastoma Sample Digital PCR Primer Probe Sensitivity Experiment

[0052] In this experiment, neuroblastoma-positive samples were used for serial dilution. Detect by the detection method given in Example 2. The results show that this system can detect the copy number ( Figure 3-Figure 4 ).

[0053] Table 2. Detection results of FZD2 gene and EFTUD2 gene of positive samples at the critical concentration.

[0054]

[0055]

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Abstract

The invention provides a primer probe for detecting amplification of a recurrence and metastasis gene FZD2 of neuroblastoma, and application of the primer probe. Primer probe sequences are a target gene upstream primer sequence FZD2-F: SEQ ID No: 1 5'-TTTCTGTCGGGCTGCTACC-3', a target gene downstream primer sequence FZD2-R: SEQ ID No: 2 5'-GTAACCGTCGGAGAAGC-3', a target gene probe sequence FZD2-P: SEQ ID No: 3 5'-FAM-GCTCCAGGAGCGCGTGGTGTGCA-BHQ1-3', an internal reference gene upstream primer sequence EFTUD2-F: SEQ ID No: 4 5'-CTCAAAGTGCGGGGACTGAT-3', an internal reference gene downstream primer sequence EFTUD2-R: SEQ ID No: 5 5'-GGCATCAGGGTGACTCCAAA-3', and an internal reference gene probe sequence EFTUD2-P: SEQ ID No: 6 5'-HEX-AGCCTGCTTCCTGGGAATGTGCTGCT-BHQ1-3'. The primer probe is used for a digital PCR kit. The kit can rapidly, specifically and sensitively detect FZD2 gene amplification, the one-step amplification method of the kit is simple, the cost is low, and the kit is very suitable for gene copy number variation conditions in various samples such as tumor tissues, circulating tumor cells and circulating tumor DNA, and is suitable for large-scale popularization and application.

Description

technical field [0001] The invention belongs to the technical field of tumor molecular detection, and in particular relates to a primer probe for detecting the amplification of neuroblastoma recurrence and metastasis gene FZD2 and its application. Background technique [0002] Neuroblastoma (GBM), which has been designated a grade IV cancer by the World Health Organization, is the most common and deadly CNS tumor in adults. Currently, standard treatment for GBM patients consists of maximal surgical resection followed by concurrent radiation and chemotherapy. Temozolomide (Temodal), a DNA alkylating agent, is the most commonly used chemotherapeutic agent. Despite these therapies, most patients eventually relapse. Therefore, there is an urgent clinical need to develop effective anti-GBM therapeutics. [0003] The prognosis of GBM patients is generally poor. GBM tumors are highly heterogeneous. On the other hand, GBM also appears to have a cellular hierarchy such that ther...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6851C12N15/11
CPCC12Q1/6886C12Q1/6851C12Q2600/118C12Q2600/166
Inventor 盛司潼张晓英黄思强朱涛
Owner SHENGZHEN CHINA GENE TECH COMPANY
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