Kit for detecting methylation of lung cancer-associated gene SHOX2 (short stature homebox2)

A technology of methylation and kit, applied in the field of early screening and detection of lung cancer, can solve the problems of poor sensitivity and specificity, and achieve the effect of improving sensitivity and specificity, high sensitivity, and easy interpretation

Inactive Publication Date: 2016-10-12
北京鑫诺美迪基因检测技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0007] Aiming at the problem of poor sensitivity and specificity of lung cancer detection products in the prior art, the purpose of the present invention is to provide a method for detecting the three target site methyl groups in the promoter

Method used

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  • Kit for detecting methylation of lung cancer-associated gene SHOX2 (short stature homebox2)
  • Kit for detecting methylation of lung cancer-associated gene SHOX2 (short stature homebox2)
  • Kit for detecting methylation of lung cancer-associated gene SHOX2 (short stature homebox2)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] Example 1 Detection test of the kit for detecting the methylation of lung cancer-related gene SHOX2

[0037] The components of the kit are as follows:

[0038]

[0039] 10 tumor plasma samples with known types were selected: 5 were positive for SHOX2 methylation, all of which were lung cancer samples; 5 were negative for SHOX2 methylation, and were non-lung cancer tumor samples.

[0040] 1. Extract free DNA from the above 10 plasma samples. The free DNA extraction kit comes from Tiangen Biochemical Technology Co., Ltd.

[0041] 2. After the extraction of free DNA is completed, bisulfite conversion is performed on the extracted DNA, and the unmethylated cytosine (C) is converted into uracil (U), while the methylated cytosine (C) remains unchanged. The purified DNA was obtained, and the transformation kit was from Tiangen Biochemical Technology Co., Ltd.

[0042] 3. Prepare PCR reaction solution: DNA polymerase, dNTPs, Mg 2+ , 10×DNA polymerase buffer, target gene 1...

Embodiment example 2

[0059] 105 lung cancer plasma samples and 15 normal plasma samples were collected from Zhejiang Cancer Hospital, and the methylation of SHOX2 gene was detected for these 120 plasma samples. The specific operation steps were as described in Example 1. The difference between the 1 / 2 / 3ct value of the target gene and the Ct value of the internal reference gene in 15 normal plasma samples was greater than 8; and for 105 lung cancer plasma samples, the 1 / 2 / 3ct value of the target gene in 90 samples was detected and the internal reference gene The differences of gene Ct values ​​were all less than 8, the sensitivity reached 85.7%, and the specificity reached 98%.

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Abstract

The invention discloses a kit for detecting methylation of a lung cancer-associated gene SHOX2 (short stature homebox2). Methylated loci of target genes in a free DNA (deoxyribonucleic acid) are detected, and the kit comprises primers corresponding to three methylated target genes and an internal reference gene primer, and preferably further comprises MGB (minor groove binder) probes corresponding to methylated loci of the three target genes, an MGB probe corresponding to an internal reference gene and MGB probes corresponding to the three methylated target genes. The detection loci comprise promoter areas of the genes and coding areas of the genes, and multi-area detection is favorable for improving the methylation accuracy and specificity of the SHOX2. The methylated loci are preferably detected more accurately by specifically combining the MGB probes and a methylation sequence. The kit is convenient to operate and easy to read, and apparatus requirements are low; a complete sealing form is adopted for the whole PCR (polymerase chain reaction) process, so that the probability of cross infection is avoided, and a result is more accurate. The kit with high detection sensitivity is applied to early screening of lung cancer.

Description

technical field [0001] The invention relates to the technical field of early screening and detection of lung cancer, in particular to a kit for detecting the methylation of lung cancer-related gene SHOX2 with cfDNA. Background technique [0002] According to the latest data, lung cancer and breast cancer rank first in the incidence of malignant tumors in men and women, respectively, and the highest number of deaths from malignant tumors in both men and women is lung cancer. Oncologists pointed out that in recent years, the incidence of cancer in my country may have shown an obvious upward trend, but less than half of the patients received standard treatment. Lung cancer is one of the common malignant tumors in my country. The age of lung cancer incidence and death in my country has risen rapidly since the age of 40, reached a peak at the age of 70, and decreased slightly after the age of 75. The age probability of onset and the number of deaths in females and males have bas...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6886C12Q1/6858C12Q2600/154C12Q2531/113C12Q2545/101C12Q2561/101
Inventor 刘沛王林海
Owner 北京鑫诺美迪基因检测技术有限公司
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