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A kit for detecting Golgi protein 73 (GP73) mRNA (messenger Ribonucleic Acid) by a fluorescent quantitative PCR (Polymerase Chain Reaction) method

A detection kit and Golgi apparatus technology, applied in the biological field, can solve the problems of fewer tumor cells, detection, and less content of tumor-specific antigens, and achieve the effects of improving detection sensitivity, providing specificity, and reducing false positive rates

Inactive Publication Date: 2019-06-11
安徽普元生物科技股份有限公司
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Problems solved by technology

However, in the early stage of tumor metastasis, there are very few tumor cells in peripheral blood, which cannot be completely relied on pathological examination. At the same time, the content of tumor-specific antigens is also very small, and it is difficult to detect them by commonly used methods such as ELISA or RIA. Therefore, The research on the gene expression of trace tumor cells in circulating blood and the detection of some specific products has aroused widespread concern

Method used

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  • A kit for detecting Golgi protein 73 (GP73) mRNA (messenger Ribonucleic Acid) by a fluorescent quantitative PCR (Polymerase Chain Reaction) method
  • A kit for detecting Golgi protein 73 (GP73) mRNA (messenger Ribonucleic Acid) by a fluorescent quantitative PCR (Polymerase Chain Reaction) method
  • A kit for detecting Golgi protein 73 (GP73) mRNA (messenger Ribonucleic Acid) by a fluorescent quantitative PCR (Polymerase Chain Reaction) method

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Experimental program
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Effect test

Embodiment 1

[0026] Fluorescent quantitative RT-PCR method to detect the expression of GP73

[0027] 1. Materials:

[0028] Trizol reagents and restriction endonucleases were purchased from Invitrogen, pGEM-T-Easy cloning system, M-MLV reverse transcriptase, Taq DNA polymerase, Oligo(dT) 15-18 Purchased from Promega Company in the United States, the common PCR instrument used for reverse transcription is Biometra PCR instrument, and the 7300 quantitative PCR instrument is a product of Applied Biosystems Company in the United States.

[0029] 2. Primer and probe design and synthesis:

[0030] Using the whole GP73 gene sequence as a template, use Primer Express v3.0 software to analyze TaqMan primers and probe sites, and consider the GP73 genomic DNA sequence to select the best combination.

[0031] The sequences of PCR primers for detection are:

[0032] The upstream primer sequence is: 5'-GACCTGAGCCAGTGCATCAA-3';

[0033] The downstream primer sequence is 5'-TCTTCTATTCGCTCCTCACACTGT-3'...

Embodiment 2

[0039] 1. Specimen testing;

[0040] Peripheral blood samples from 6 patients with primary liver cancer diagnosed by the disease, the peripheral blood nucleated cells were separated, the cells were collected by centrifugation and the total RNA was extracted with Trizol reagent, 1ul RNA was taken, and oligo(dT) was used in a total volume of 25ul 15-18 After performing reverse transcription as primers, use the upstream and downstream primers of the standard to perform PCR amplification on the 7300 PCR instrument of ABI Company. 40 cycles of amplification were performed, with a final extension at 72°C for 10 minutes and then set at 4°C. At the same time, add standard substance to test for standard curve. After the measurement results were processed by the instrument, the expression level of GP73 in the test specimen was calculated according to the standard curve.

[0041] 2. Results

[0042](1) Preparation of standard products:

[0043] After sequencing, the above designed st...

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Abstract

The invention relates to a kit for detecting Golgi protein 73 (GP73) mRNA (messenger Ribonucleic Acid) by real-time fluorescent quantitative PCR (Polymerase Chain Reaction), cDNA (complementary Deoxyribonucleic Acid) is obtained by reverse transcription (RT) of an mRNA sample, and the mRNA expression quantity of a specimen GP73 can be quantitatively detected by combining a real-time fluorescent quantitative PCR detection technology. The kit provided by the invention can be used for detecting the expression of GP73 in peripheral blood of a patient in clinic and scientific research, and providesan important reference basis for determination and prognosis judgment of primary liver cancer (PHC).

Description

technical field [0001] The invention belongs to the field of biological technology, and is a reagent that can accurately and quantitatively detect the expression of human Golgi protein 73 (GP73) mRNA in a specimen by obtaining cDNA through reverse transcription (RT) mRNA samples, combined with real-time fluorescent quantitative PCR detection technology box. Background technique [0002] The invention belongs to the field of biological technology, and is a kit for obtaining cDNA by reverse transcription (RT) mRNA samples, combined with real-time fluorescent quantitative PCR detection technology, which can accurately and quantitatively detect the expression level of human GP73 mRNA in samples. [0003] Cancer is a major disease that seriously threatens human life and social development. It is the first cause of death in cities and the second cause of death in rural areas. Using scientific methods to prevent and control cancer has become one of the most important public health...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/686
Inventor 不公告发明人
Owner 安徽普元生物科技股份有限公司
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