Primer and probe for early detecting methylation of lung cancer gene and reagent kit

An early detection and methylation technology, which is applied in the detection/testing of protozoa, microorganisms, biochemical equipment and methods, etc., can solve the problems of high reagent cost, cumbersome and inconvenient experimental operation, and reduce the specificity of detection, so as to achieve specificity. Advantages, fewer types of reagents, and the effect of saving reagent costs

Active Publication Date: 2019-09-13
SICHUAN ORIENTER BIOLOGICAL TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

These methods jointly detect a large number of genes. Although it can increase the detection rate of lung cancer, it will also reduce the specificity of detection. At the same time, multi-gene detection is cumbersome and inconvenient in experimental operation, and the cost of reagents is high.

Method used

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  • Primer and probe for early detecting methylation of lung cancer gene and reagent kit
  • Primer and probe for early detecting methylation of lung cancer gene and reagent kit
  • Primer and probe for early detecting methylation of lung cancer gene and reagent kit

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] This embodiment provides the description information of fluorescent reporter dyes and fluorescent quencher dyes labeled with each detection probe, specifically as follows:

[0040] When CDO1 is detected alone, the primer pair used to detect CDO1 gene methylation is the sequence shown in SEQ ID No: 1 and SEQ ID No: 2, and the probe is HEX-TTTGCGACGATATTTTTACGTTTCGGTATTT-BHQ1; the primer pair used to detect the internal reference ACTB is The sequence shown in SEQ ID No: 4 and SEQ ID No: 5, the probe is FAM-ATATTGGTTCGTGTGATAAGGTTATGAGGTTG-BHQ1.

[0041] When CDO1, RASSF1A and SHOX2 are jointly detected, the primer pair used to detect CDO1 gene methylation is the sequence shown in SEQ ID No: 1 and SEQ ID No: 2, and the probe is HEX-TTTGCGACGATATTTTTACGTTTCGGTATTT-BHQ1; used to detect the internal reference ACTB The primer pair is the sequence shown in SEQ ID No: 4 and SEQ ID No: 5, and the probe is FAM-ATATTGGTTCGTGTGATAAGGTTATGAGGTTG-BHQ1; the primer pair for detecting RA...

Embodiment 2

[0043] This embodiment provides PCR amplification reaction reagents, positive control substance and negative control substance containing the primer pair and probe described in Example 1:

[0044] 1. PCR amplification reaction reagents: the final concentration of each primer of the primer pair is 200nM, the final concentration of the probe is 150nM, the DNA polymerase is 1U, the magnesium ion concentration is 2mM, and the dNTPs is 100uM.

[0045] 2. Positive control substance: the concentration is 10ng / ul, and the genomic DNA of the lung cancer cell line MSTO-211H is mixed with the genomic DNA of healthy people according to the ratio of 1:9;

[0046] 3. Negative control substance: healthy human genome DNA at a concentration of 10ng / ul.

Embodiment 3

[0048] This example is a method for early detection of lung cancer using the PCR amplification reaction reagent described in Example 2.

[0049] 1. Collection of samples

[0050] 1.1 Collection of negative samples: 20 cases of alveolar lavage fluid samples were taken from healthy people aged 20-35 without family history of lung cancer or lung cancer disease history.

[0051] 1.2 Collection of samples from lung cancer patients: 67 cases of alveolar lavage fluid samples clinically identified as lung cancer patients were collected, including 31 males and 36 females, with an age range of 45-87 years and a median age of 62 years; There were 19 cases of carcinoma, 23 cases of adenocarcinoma, 21 cases of bronchioloalveolar carcinoma, and 4 cases of small cell lung cancer.

[0052] 2. Extraction of DNA from alveolar lavage fluid samples

[0053] 2.1 Put the fresh alveolar lavage fluid into a 50ml centrifuge tube, centrifuge at 3000rpm for 5-10 minutes, discard the supernatant, and k...

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Abstract

The invention discloses a primer and probe for early detecting methylation of a lung cancer gene. The primer and probe comprise a primer pair and a probe for detecting methylation of a CDO1 gene, wherein the primer pair has the sequences as shown in SEQID No:1 and SEQID No:2, and the probe has a sequence as shown in SEQID No:3. The clinical detection sensibility of the primer and probe is greaterthan 77.6%, and the specificity is greater than 98%. The invention also discloses a reagent kit based on the primer and probe. The reagent kit is simple to operate and low in reagent cost.

Description

technical field [0001] The invention relates to the field of gene detection, in particular to primers, probes and kits for early detection of lung cancer gene methylation. Background technique [0002] Lung cancer (lung cancer) is the abbreviation of primary bronchial lung cancer, and it is the first clinical common malignant tumor. According to "Cancer Statistics in China, 2015", there are more than 600,000 new lung cancer patients in my country every year, and more than 500,000 deaths, while the overall five-year survival rate is only 16.1%, which is the malignant tumor with the highest mortality rate. About 71% of lung cancer patients are men and 29% are women. Lung cancer is mainly divided into non-small cell lung cancer and small cell lung cancer, and non-small cell lung cancer is divided into squamous cell carcinoma, adenocarcinoma, large cell carcinoma, etc. In my country, patients with non-small cell lung cancer account for about 85% of the total number of lung canc...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12N1/11
CPCC12Q1/6886C12Q2600/154
Inventor 刘亚宝刘春晖李佳
Owner SICHUAN ORIENTER BIOLOGICAL TECH
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