A kit for detecting human cytokeratin 7 through one-step real-time fluorescence quantitative reverse transcription polymerase chain reaction

A technology of reverse transcription polymerase and real-time fluorescence quantification, which is applied in the biological field, can solve the problem of small number of cells, and achieve the effects of reducing false positive interference, avoiding cross-contamination, and improving specificity

Inactive Publication Date: 2019-01-15
安徽普元生物科技股份有限公司
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  • Application Information

AI Technical Summary

Problems solved by technology

Surviving tumor cells in the blood circulation are the basis of distant metastasis and recurrence. Although various detection methods for various colorectal cancers continue to increase, the number of such cells is extremely small, and it is not easy to be detected by clinical routine methods

Method used

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  • A kit for detecting human cytokeratin 7 through one-step real-time fluorescence quantitative reverse transcription polymerase chain reaction
  • A kit for detecting human cytokeratin 7 through one-step real-time fluorescence quantitative reverse transcription polymerase chain reaction
  • A kit for detecting human cytokeratin 7 through one-step real-time fluorescence quantitative reverse transcription polymerase chain reaction

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] One-step fluorescence quantitative RT-PCR method was used to detect the expression of CK7.

[0024] 1. Materials:

[0025] Trizol reagents and restriction endonucleases were purchased from American Invitrogen Company, pGEM-T-Easy cloning vector, M-MLV reverse transcriptase, Taq DNA polymerase, Oligo(dT)15-18 were purchased from American Promega Company, type ABI7500 The quantitative PCR instrument is a product of ABI Company in the United States.

[0026] 2. Primer and probe design and synthesis:

[0027] Using the CK7 full-length cDNA sequence (GenBank accession number NM_031144.3) as a template, use Primer ExpressTM (V3.0, American ABI Company) software to analyze the TaqMan primer and probe sites, and according to the CK7 genomic DNA sequence, from it Choose the best combination. The primers of the standard product of the present invention and the primers for inspection are the same primers.

[0028] The upstream primer sequence is

[0029] 5'-ACCAGTTCGCCATGGATG...

Embodiment 2

[0038] Application of fluorescent quantitative RT-PCR method to detect CK7.

[0039] Peripheral blood samples from 8 patients with malignant tumors confirmed by pathology were separated. Peripheral blood nucleated cells were collected by centrifugation after washing with PBS. Total RNA was extracted with Trizol reagent, and 2.5 μl RNA was taken in a volume of 50 μl reverse transcription polymerase chain reaction. , use the upstream and downstream primers to carry out PCR amplification on the ABI 7500 PCR instrument, the conditions are 45°C reverse transcription for 30 minutes, 95°C pre-denaturation for 5 minutes, 95°C for 30 seconds, 60°C for 30 seconds, 72°C for 30 seconds, a total of 40 Cycle, 72°C extension for 10 minutes followed by 4°C. At the same time, add standard substance to test for standard curve. The measurement results were processed by the instrument and calculated according to the standard curve to calculate the CK7 expression level of the test specimen.

[004...

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Abstract

The invention relates to a kit for detecting human cytokeratin 7 through one-step real-time fluorescence quantitative reverse transcription polymerase chain reaction. The kit adopts mRNA as a template, RT-PCR and real-time fluorescence quantitative PCR can be performed in the same reaction system without opening a tube cover, avoiding cross-contamination, and the mRNA expression of human cytokeratin 7 (CK7) in the sample can be quantitatively and precisely detected. The kit can be used to detect the expression of CK7 in peripheral blood, lymph nodes and other specimens of patients in clinicaland scientific research, to assist diagnosis, guide the treatment of colorectal cancer and predict the prognosis.

Description

technical field [0001] The invention belongs to the field of biological technology, and is a detection technology that uses mRNA as a template to carry out reverse transcription and real-time fluorescent quantitative PCR in the same reaction system, and can accurately and quantitatively detect human cytokeratin 7 (CK7) in a sample Kit for mRNA expression. Background technique [0002] In the middle of the last century, with the continuous change and improvement of people's living environment, eating habits and living standards, the morbidity / death rate of various tumors also showed an upward trend year by year. The morbidity and mortality of colorectal cancer are second only to lung and breast cancer, and it has become the third malignant tumor. Colorectal cancer has highly malignant and aggressive biological characteristics, and often infiltrates and metastasizes. This biological characteristic is an important factor leading to poor prognosis and high mortality in patients...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6851
CPCC12Q1/6851C12Q1/6886C12Q2600/166C12Q2561/113C12Q2563/107C12Q2521/107C12Q2545/101
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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