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Kit for detecting methylation level of lung cancer associated SHOX2 gene promoter region

A gene promoter region and kit technology, applied in the biological field, can solve the problems of high cost of genome-wide methylation analysis and is not suitable for clinical diagnosis, and achieve the effect of low instrument requirements, uncomplicated process, and high sensitivity

Active Publication Date: 2016-02-10
湖南宏雅基因技术有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, the cost of genome-wide methylation analysis is too high to be suitable for clinical diagnosis

Method used

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  • Kit for detecting methylation level of lung cancer associated SHOX2 gene promoter region
  • Kit for detecting methylation level of lung cancer associated SHOX2 gene promoter region

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Embodiment 1 SHOX2 gene methylation quantitative detection method:

[0041] ① Process the sample to be tested. The nucleic acid sample is derived from: nucleic acid extracted from a sample containing cells, such as alveolar lavage fluid, tissue taken from a lesion, pleural effusion, sputum, etc.; or nucleic acid containing nucleic acid derived from cells Samples, such as plasma, serum, etc., to obtain sample templates;

[0042] A, extract DNA (Qiagen company) from the sample to be tested;

[0043] B, carry out bisulfite modification (Qiagen company) to the extracted DNA;

[0044] C. Purifying the modified DNA to obtain a sample template.

[0045] ② Design and synthesize SHOX2 gene amplification primers, internal reference gene ACTB amplification primers, Taqman probes, and blocking agents (such as the former SEQ ID NO.1-SEQ ID NO.8);

[0046] ③ Real-time fluorescent quantitative PCR detection, the reaction conditions of real-time fluorescent quantitative PCR detectio...

Embodiment 2

[0059] Example 2 Analysis of methylation degree of SHOX2 gene in different lung cancer samples

[0060] Collect 80 lung cancer tissue samples from a tertiary hospital in Changsha, and collect 21 normal lung cancer tissue samples at the same time, use the kit to extract gDNA, and then treat the samples with bisulfite. Then the above kit and system are used to detect the SHOX2 gene and the internal reference gene GAPDH in the cancer tissue sample and the normal tissue sample. In normal tissue samples, the difference between the Cp value of the SHOX2 gene and the Cp value of the internal control gene was more than 7, while in tumor tissue samples, the difference between the Cp value of the SHOX2 gene and the Cp value of the internal control gene was detected in 61 cases. Methylation occurred in lung cancer tissue samples, the sensitivity reached 76.3%, and the specificity reached 96%.

[0061]

[0062]

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Abstract

The invention discloses a Kit for detecting the methylation level of a lung cancer associated SHOX2 gene promoter region, which comprises a target gene primer pair, a reference gene primer pair, Taqman probes, and blocking agents, wherein the Taqman probe is used for detecting target genes and reference genes, and the blocking agent has a blocking effect. The target gene primer pair comprises a target gene forward primer shown in a sequence SEQ ID NO.1 and a target gene reverse primer shown in a sequence SEQ ID NO.2; and the reference gene primer pair comprises a reference gene forward primer shown in a sequence SEQ ID NO.3 and a reference gene reverse primer shown in a sequence SEQ ID NO.4. The target gene Taqman probe for detecting is shown in a sequence SEQ ID NO.5, and the reference gene Taqman probe is shown in a sequence SEQ ID NO.6. The blocking agents are shown in a sequence SEQ ID NO.7 and a sequence SEQ ID NO.8.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a kit for detecting the methylation degree of the promoter region of the SHOX2 gene related to lung cancer. Background technique [0002] Lung cancer is currently one of the malignant tumors with the highest incidence in China, and its mortality rate is also high. The prognosis of the quality of life is not optimistic, and its five-year survival rate is only 17%. In clinical practice, early diagnosis of lung cancer has always been difficult, but early detection is crucial to the effective treatment of cancer patients. At present, histological and cytological examinations after the appearance of clinical symptoms are the gold standard for the diagnosis of lung cancer. However, early lung cancer is hardly noticed by doctors and patients because it often has no special symptoms, and it is difficult to detect and diagnose early with conventional diagnostic methods. , and the...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
Inventor 贺毅憬彭艳肖飞刘杨虞健涂超峰
Owner 湖南宏雅基因技术有限公司
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