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Primers and probe for quantitatively detecting human VSTM1 (Visual Short Term Memory 1) gene expression level in real time

A probe and primer pair technology, applied in the field of primers and probes for quantitatively detecting the expression level of human VSTM1 gene in real time, can solve the problems that the general significance of tumor suppressor genes cannot be proved, and the gene function is unknown.

Inactive Publication Date: 2012-08-29
PEOPLES HOSPITAL PEKING UNIV
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Problems solved by technology

The current problem is that it is not yet possible to prove the general significance of the discovered tumor suppressor genes in the pathogenesis of AML, and with the successful completion of the Human Genome Project, it is possible to find more diagnostic and drug targets in most human leukemias. point
Although the entire human genome has been sequenced, the functions of many genes remain largely unknown

Method used

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  • Primers and probe for quantitatively detecting human VSTM1 (Visual Short Term Memory 1) gene expression level in real time
  • Primers and probe for quantitatively detecting human VSTM1 (Visual Short Term Memory 1) gene expression level in real time
  • Primers and probe for quantitatively detecting human VSTM1 (Visual Short Term Memory 1) gene expression level in real time

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Embodiment 1, the design of specific primer pair and probe

[0032] Design specific primer pair A (composed of upstream primer VSTM1-FP and downstream primer VSTM1-RP, target sequence is 137bp) and probe A (probe VSTM1- probe):

[0033] Upstream primer VSTM1-FP (sequence 1 of the sequence listing): 5'-GCCGAGGCAGATTTATCCAA-3';

[0034] Downstream primer VSTM1-RP (sequence 2 of the sequence listing): 5'-CCTGGGTGGTGTCTGAAGCT-3';

[0035] Probe VSTM1-probe (5'→3'):

[0036] FAM-CTCGACGGCAGACCCCCAAGG-BHQ (the nucleotide sequence is sequence 3 in the sequence listing).

[0037] Design specific primer pair B (composed of upstream primer ABL1-F and downstream primer ABL1-R, target sequence is 124bp) and probe B (probe ABL1-T-probe):

[0038] Upstream primer ABL1-F (sequence 4 of the sequence listing): 5'-TGGAGATAACACTCTTAAGCATAACTAAAGGT-3';

[0039] Downstream primer ABL1-R (sequence 5 of the sequence listing): 5'-GATGTAGTTGCTTGGGACCCA-3';

[0040] Probe ABL1-T-probe (5'...

Embodiment 2

[0045] Embodiment 2, the preparation of relevant plasmid

[0046] 1. Preparation of positive control plasmid (plasmid containing VSTM1 gene fragment)

[0047] Using the cDNA of bone marrow mononuclear cells of normal people (volunteers) with positive VSTM1 gene expression as a template, PCR amplification was performed to obtain PCR amplification products.

[0048] The primer pairs used in PCR amplification are as follows:

[0049] Upstream primer: 5'-GTGAGAAGGAAACTGCAAGAGTGGG-3';

[0050] Downstream primer: 5'-TTGATATGGACGAAGAGCAAGGAAA-3'.

[0051] Sequencing results show that the nucleotide sequence of the PCR amplification product is shown as sequence 7 in the sequence listing (993bp).

[0052] Insert the PCR amplification product into the pMD18-T plasmid (pMD18-T vector system, Shanghai Haojia Company, catalog number: D101A) to obtain a positive control plasmid (the backbone plasmid is the pMD18-T plasmid, inserted at the ECOR V site VSTM1 gene fragment shown in SEQ ID ...

Embodiment 3

[0060] Embodiment 3, detect the sensitivity detection of positive control plasmid

[0061] The positive control plasmid obtained in Example 2 was diluted 10 times with sterilized double distilled water for injection to obtain each dilution (each microliter contained 10 7 、10 6 、10 5 、10 4 、10 3 、10 2 、10 1 、10 0 copy VSTM1 gene fragment); the internal reference control plasmid obtained in Example 2 was diluted 10 times with sterilized double distilled water for injection to obtain each dilution (each microliter contained 10 6 、10 5 、10 4 、10 3 、10 2 、10 1 、10 0 copies of the ABL gene fragment); the copy number of the VSTM1 gene fragment or the ABL gene fragment was calculated by measuring the absorbance value.

[0062] Each positive control plasmid dilution was carried out RQ-PCR on a fluorescence real-time quantitative PCR instrument (7500-FAST type of American ABI Company) using the specific primer pair A and probe A obtained in Example 1. Each internal control...

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Abstract

The invention discloses primers and a probe for quantitatively detecting human VSTM1 (Visual Short Term Memory 1) gene expression level in real time. The invention provides a kit comprising a first specific primer pair and a first probe, wherein the first specific primer pair is a primer pair comprising a DNA (Deoxyribose Nucleic Acid) fragment as showed in a sequence 1 in a sequence table and a DNA fragment as shown in a sequence 2 in the sequence table, and the nucleotide sequence of the first probe is as shown in a sequence 3 in the sequence table. The kit has the following function (a), (b), (c) or (d): (a) auxiliary diagnosis of tumor patients; (b) auxiliary evaluation of the treatment effects of the tumor patients; (c) auxiliary identification of tumor cell systems; and (d) quantitative detection of the VSTM1 gene expression level. The primer pair and the probe can be used for quantitatively detecting the VSTM1 gene expression level, so that the kit can be applied to the auxiliary identification of the blood tumor cell systems or the auxiliary diagnosis of the blood tumor patients (particularly leukemia patients). Therefore, the kit can play an important role in the field ofmedical detection.

Description

technical field [0001] The invention relates to a primer and a probe for real-time quantitative detection of human VSTM1 gene expression level. Background technique [0002] Leukemia is a heterogeneous hematopoietic system tumor with malignant lesions of hematopoietic stem / progenitor cells. The most common malignant tumor in adolescents, the pathogenesis is still unclear. [0003] Advances in molecular biology have proved that genetic abnormalities are the basis of leukemia formation. The diagnostic criteria for leukemia and lymphoma released by WHO in 2008 emphasized the importance of genetic abnormalities in the diagnosis, stratification and prognosis of hematological malignancies. Oncogene activation caused by acute myeloid leukemia (AML)-related chromosomal translocations, such as AML1 / ETO, PML / RARα, CBFb / MYH11, etc., has been recognized and used in clinical diagnosis, targeted therapy, etc. play an important role in. But activation of cellular oncogenes represents on...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
Inventor 阮国瑞黄晓军谢敏马大龙韩文玲陈珊珊李金兰冷馨
Owner PEOPLES HOSPITAL PEKING UNIV
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