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Primer probe set, kit and method for quantitatively detecting expression amount of GNA15 gene

A technology for quantitative detection of gene expression, applied in the field of biochemical detection, can solve the problem of tumor cells in the blank stage, and achieve the effects of high reliability, accurate results and high sensitivity

Pending Publication Date: 2020-05-05
THE FIRST AFFILIATED HOSPITAL OF ZHENGZHOU UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is no relevant report on the detection of GNA15 gene expression, that is, the use of GNA15 gene expression to assist in the identification of tumor cells is still in the blank stage

Method used

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  • Primer probe set, kit and method for quantitatively detecting expression amount of GNA15 gene
  • Primer probe set, kit and method for quantitatively detecting expression amount of GNA15 gene
  • Primer probe set, kit and method for quantitatively detecting expression amount of GNA15 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Example 1 Primer probe set for quantitative detection of GNA15 gene expression according to the present invention

[0027] The primer probe set of the present invention includes component A and component B, and the component A includes: upstream primer GNA15-FP: 5'-GAGAGCCTCGCATTGTTTGG-3' (ie, the nucleotide sequence shown in SEQ ID NO. 1) , downstream primer GNA15-RP: 5'-GGATGTCGGTTTTGTTGAGAAAG-3' (ie nucleotide sequence shown in SEQ ID NO.2), probe GNA15-probe: FAM-TACCCTGGTTCAAAAGCACATCCGTCAT-BHQ (ie nuclear shown in SEQ ID NO.3) nucleotide sequence), the 5' end of the probe GNA15-probe is labeled with a FAM luminescent group, and the 3' end is labeled with a fluorescence quenching group BHQ;

[0028] The component B includes: the upstream primer ABL1-FP: 5'-TGGAGATAACACTCTAAGCATAACTAAAGGT-3' (that is, the nucleotide sequence shown in SEQID NO. 4), the downstream primer ABL1-RP: 5'-GATGTAGTTGCTTGGGACCCA-3' (that is, SEQID NO.4) The nucleotide sequence shown in ID NO...

Embodiment 2

[0029] Example 2 Kit for quantitative detection of GNA15 gene expression according to the present invention

[0030] The kit of the present invention includes component A, component B, positive control plasmid and internal reference control plasmid in Example 1; in component A: upstream primer GNA15-FP: downstream primer GNA15-RP: probe GNA15-probe The molar ratio is 3:3:2; in component B: the molar ratio of the upstream primer ABL1-FP, the downstream primer ABL1-RP and the probe ABL1-probe is 3:3:2;

[0031] The positive control plasmid is a positive plasmid inserted into the GNA15 gene fragment shown in SEQ ID NO.7 at the XhoI / KpnI restriction site of the GV219 plasmid;

[0032] The internal reference control plasmid is the internal reference plasmid with the ABL1 gene fragment shown in SEQ ID NO. 8 inserted into the XhoI / KpnI restriction site of the GV219 plasmid.

Embodiment 3

[0033] Example 3 The sensitivity detection of the positive control plasmid and the internal reference control plasmid of the present invention and the drawing of the fluorescence standard curve specifically include the following contents:

[0034] The first step is to dilute the positive control plasmid in Example 2 by 10-fold gradient with sterile double-distilled water for injection to obtain positive control plasmid solutions containing different copy numbers of GNA15 gene fragments: 10 6 , 10 5 , 10 4 , 10 3 , 10 2 copies / μL;

[0035] Carry out a 10-fold gradient dilution of the internal reference control plasmid in Example 2 with sterile double-distilled water for injection to obtain an internal reference control plasmid solution containing different copy numbers of ABL1 gene fragments: 10 6 , 10 5 , 10 4 , 10 3 , 10 2 copies / microliter;

[0036] In the second step, PCR reactions are performed on the positive control plasmid solution and the internal reference co...

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Abstract

The present invention discloses a primer probe set, a kit and a method for quantitatively detecting expression amount of GNA15 gene. The primer probe set includes components A and components B, the components A comprise an upstream primer GNA15-FP, a downstream primer GNA15-RP and a probe GNA15-probe, and the components B comprise an upstream primer ABL1-FP, a downstream primer ABL1-RP and a probeABL1-probe. Advantages are as follows: the method can quantitatively detect the relative expression amount of the GNA15 gene in cells, has sensitivity of GNA15 and sensitivity of ABL1 both up to 100copies, is high in sensitivity, accurate in results, and high in reliability, provides a new auxiliary diagnostic method or an auxiliary identification method for hematologic tumor cells (especially acute myelogenous leukemia and acute lymphoblastic leukemia) and has relatively great application prospects.

Description

technical field [0001] The invention relates to the field of biochemical detection, in particular to a primer-probe set for quantitatively detecting GNA15 gene expression, a kit comprising the primer-probe set, and a quantitatively detecting GNA15 gene expression Methods. Background technique [0002] GNA15 is a member of the GNA gene family, which includes GNAQ, GNA11, GNA14, and GNA15. The protein expressed by GNA15 is GNA15 protein (ie Gα15) belonging to the Gαq subfamily, expressed in highly specific cell types, such as hematopoietic stem cells and epithelial cells at specific stages of differentiation, and can participate in regulating cell apoptosis or proliferation, such as Gα15 Promotes cell proliferation and inhibits cell apoptosis by coupling to a wide range of GPCRs. Human GNA15 gene is located at 19p13.3. GNA15 gene may play an oncogene role in a variety of solid tumors such as gastroenteropancreatic neuroendocrine tumors, liver cancer, and ovarian cancer. NFκ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6851C12Q1/6886C12N15/11
CPCC12Q1/6851C12Q1/6886C12Q2600/158C12Q2600/166C12Q2531/113C12Q2545/114C12Q2563/107
Inventor 王冲王树娟刘延方姜中兴张雨李梦亚范熠李涛李亚飞
Owner THE FIRST AFFILIATED HOSPITAL OF ZHENGZHOU UNIV
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