Primers, probe, and kit for detecting BRCA1 gene expression

A technology of gene expression and kit, which is applied in the field of biotechnology and clinical molecular diagnosis, can solve the problems of limiting the scope of application of BRCA1 expression, high cost and price of double-probe hybridization method, and high subjectivity of test results, so as to achieve credible test results, High sensitivity and specificity, good accuracy

Inactive Publication Date: 2016-03-23
武汉海吉力生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the experimental process of immunohistochemical method is cumbersome, requires a wide variety of reagents, takes a long time, and the test results are highly subjective; the SYBRGreenⅠ method in fluorescent quantitative PCR has poor specificity, while the double-probe hybridization method has a high cost
The defects of the above-mentioned prior art limit the scope of application of BRCA1 expression detection

Method used

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  • Primers, probe, and kit for detecting BRCA1 gene expression
  • Primers, probe, and kit for detecting BRCA1 gene expression
  • Primers, probe, and kit for detecting BRCA1 gene expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Embodiment 1: the preparation of kit of the present invention

[0042] The kit of the present invention consists of the following:

[0043] (1) The preparation of the first-strand cDNA synthesis system is shown in Table 1.

[0044] Table 1

[0045]

[0046]

[0047] (2) The preparation of fluorescent quantitative PCR reaction solution is shown in Table 2.

[0048] Table 2

[0049] Reagent

Dosage

Premix (Self-configured)

1.5μL

BRCA1 gene upstream and downstream primer pair, B2M gene upstream and downstream primer pair

1~10pM for each pair

BRCA1 gene Taqman fluorescent probe, B2M gene Taqman fluorescent probe

1~10pM each

Add DEPC water to

20 μL

[0050] Note: Main components of Premix: DNA polymerase 1~2U, 2.0~5.0mMMgCl 2 , 0.2~0.8mMdNTPs, 10×Buffer, RNaseH (ribonuclease H). Premix is ​​a self-prepared premix to adapt to BRCA1 and B2M primer probes, which can quickly and accurately detect and quantif...

Embodiment 2

[0052] Embodiment 2: Detect the expression level of BRCA1mRNA with the kit prepared in embodiment 1

[0053] In this example, RNA was extracted from paraffin-embedded tissue sections of 60 clinical breast cancer patients in our company, and was quantified as a template for PCR detection. For specific operation steps, refer to the QIAGENRNeasyFFPEKit Paraffin-Embedded Tissue RNA Extraction Kit Manual Extraction Kit to extract sample RNA. The purity and concentration of the extracted RNA were calculated by ultraviolet spectrophotometry, and the extracted RNA was diluted to the same concentration with 0.1% DEPC water, and the concentration range was 10 ng / μL.

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Abstract

The invention discloses primers, a probe and a kit for detecting the BRCA1 gene expression. The nucleotide sequences of upstream and downstream primers of BRCA1 gene are respectively represented by the SEQ ID No. 1-2. The nucleotide sequence of Taqman fluorescence probe of BRCA1 gene is represented by the SEQ ID No.3. The invention also discloses a detection kit composed of the primers and probe mentioned above, and an application method of the kit. The provided kit can detect samples in different forms such as fresh frozen tissues, paraffin embedded samples, and the like, and has the advantages of high sensitivity, high specificity, and wide application range. The kit comprises premix, a sample can be added directly, a sample does not need to be mixed with enzyme, the operation is convenient and simple, the judgment method becomes more scientific and reasonable, the error generated during the experiment process is reduced, the precision is high, furthermore, the detection speed of the detection kit is quick, and the detection result can be obtained within two hours.

Description

technical field [0001] The invention relates to the technical fields of biotechnology and clinical molecular diagnosis, in particular to a primer, a probe and a kit for rapidly and efficiently detecting the expression level of human BRCA1 gene mRNA by using a fluorescent quantitative PCR method. Background technique [0002] Breast cancer is the second most common malignant tumor in the world, ranking first among female malignant tumors. And the incidence rate showed a linear upward trend. 5-10% of breast cancers are caused by mutations in breast cancer susceptibility genes with high penetrance. One of the most common susceptibility genes is the BRCA1 gene. [0003] The BRCA1 (breast cancers susceptibility gene 1) gene is located at 17q21, about 81kb, and contains 23 exons. BRCA1 is a tumor suppressor gene with autosomal dominant inheritance. The expressed protein contains 1863 amino acids and has important biological activities. It is involved in cell cycle regulation, D...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6886C12Q1/6851C12Q2600/106C12Q2600/158C12Q2531/113C12Q2561/101C12Q2545/113
Inventor 王秀娟周艳琳段卫涛赵平锋
Owner 武汉海吉力生物科技有限公司
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