Application of hoxa9 methylation detection reagent in the preparation of lung cancer diagnostic reagent

A HOXA9 and detection reagent technology, applied in the field of genetic diagnosis, can solve the problems of limitation, patient loss, false positive rate, etc., and achieve the effects of high sensitivity, convenient sampling process, and wide application range

Active Publication Date: 2021-10-22
CREATIVE BIOSCIENCES (GUANGZHOU) CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

(4) Bone scan: the sensitivity of detection of bone metastases of lung cancer is high, but there is a certain false positive rate
If an early tumor screening product cannot screen out all or most of the patients, those who miss the test will not be able to receive sufficient risk warnings, thus delaying the timing of treatment, which is a huge loss for patients loss
[0018] Although some tumor markers related to lung cancer have been found in the prior art, the sensitivity and specificity of these tumor markers cannot meet the demand due to the limitation of detection reagents or detection methods for these tumor markers. Therefore, the current There is still a need for further research in the field of screening methods that can actually be applied to lung cancer
However, although non-invasive screening has unique advantages in sampling, it also has some limitations in other aspects, such as adenocarcinoma in lung cancer, which is difficult to cough up through sputum due to exfoliated cells in the deep lung Generally speaking, those skilled in the art would think that this type of lung cancer is not suitable for non-invasive screening
On the other hand, even for other types of lung cancer, the currently reported non-invasive screening methods are difficult to meet the requirements of clinical use
Although relevant research has been progressing for many years, there is still no non-invasive screening method for lung cancer that can be promoted to the clinic

Method used

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  • Application of hoxa9 methylation detection reagent in the preparation of lung cancer diagnostic reagent
  • Application of hoxa9 methylation detection reagent in the preparation of lung cancer diagnostic reagent
  • Application of hoxa9 methylation detection reagent in the preparation of lung cancer diagnostic reagent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0073] Example 1: Selection of Detection Target Genes

[0074] Using methylated DNA as the detection target has obvious advantages. Compared with protein markers, DNA can be amplified and easily detected; compared with mutation markers, DNA methylation sites are located in Specific parts of the gene, generally in the promoter region, which makes detection easier and more convenient. In order to complete the present invention, the inventors selected HOXA9, SHOX2, PCDHGA12, HOXD8, and GATA3 from hundreds of candidate genes through multiple rounds of screening as candidate detection genes, and the β-actin gene as an internal reference gene. The distribution of gene methylation sites, the primers and probes designed for detection were used for real-time fluorescent quantitative methylation-specific polymerase chain reaction (real-time fluorescentquantitative methylation-specific PCR, qMSP) detection. The detection primers and probes for each gene are as follows:

[0075] The det...

Embodiment 2

[0124] Example 2: Detection of HOXA9, SHOX2, PCDHGA12, HOXD8 and GATA3 genes in sputum

[0125] Sample information: A total of 90 cases of sputum samples were tested, including 55 cases of normal control group samples, 35 cases of cancer group control samples, 12 cases of squamous cell carcinoma, 6 cases of small cell carcinoma, 9 cases of adenocarcinoma, and large There were 2 cases of cell carcinoma and 6 cases of unclassified lung cancer.

[0126] Experimental procedure:

[0127] a. Collect sputum specimens from patients diagnosed with lung cancer and non-lung cancer patients. After thickening with DTT, centrifuge to separate the pellet and separate the cells, wash them twice with PBS, and then use Magen’s DNA extraction kit (HiPure FFPE DNA Kit, D3126-03) to extract DNA.

[0128] b. Use the DNA conversion kit (EZ DNA Methylation Kit, D5002) of ZYMO RESEARCH Biological Company to carry out the bisulfite modification of DNA.

[0129] c. The liquid distribution system is a...

Embodiment 3

[0139] Example 3: Detection of HOXA9 and SHOX2 genes in sputum

[0140] A large number of documents show that SHOX2 can be used as a marker for the detection of lung cancer, and there are patents [CN201510203539-method and kit for diagnosing methylation of human SHOX2 gene and human RASSF1A gene-application disclosure], SHOX2 in alveolar lavage fluid, lesion sites It has a high detection rate in samples such as tissue, pleural effusion, and sputum. In order to verify the detection effect of HOXA9, in this example, the detection efficiency of the SHOX2 gene uses the primer and probe sequences disclosed in the patent CN201510203539, and the SHOX2 gene is expressed as SHOX2_n3 to distinguish it from the ones in Examples 1 and 2 of the present invention. The SHOX2 gene detected by self-designed primers and probes.

[0141] The detection primers and probes for each gene are as follows:

[0142] The detection primers and probes for HOXA9 are:

[0143] SEQ ID NO: 1HOXA9-F2 Primer ...

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Abstract

The invention belongs to the field of gene diagnosis, and relates to the application of a detection reagent with HOXA9 methylation as a detection object in the preparation of a lung cancer diagnostic reagent. The present invention uses HOXA9 as a tumor marker for detecting lung cancer. Its sensitivity in sputum is as high as 74.3%, and its specificity is as high as 95%. The detection sensitivity is higher than the existing reported lung cancer tumor markers. The sensitivity is as high as 61.9%, and the specificity is as high as 95%. Especially for adenocarcinoma, the sensitivity has been greatly improved, and it has great application value for the detection and diagnosis of adenocarcinoma.

Description

technical field [0001] The invention belongs to the field of gene diagnosis, and more specifically, the invention relates to the application of a detection reagent for diagnosing human HOXA9 gene methylation in the preparation of lung cancer diagnostic reagents, and a method for detecting human HOXA9 gene methylation. [0002] technical background [0003] Lung cancer is a malignant tumor of the lung originating from the bronchial mucosa, glands or alveolar epithelium. According to the pathological type, it can be divided into: 1. Small cell lung cancer (SCLC): a special pathological type of lung cancer, with obvious tendency of distant metastasis and poor prognosis, but most patients are sensitive to radiotherapy and chemotherapy. 2. Non-small cell lung cancer (NSCLC): other pathological types of lung cancer other than small cell lung cancer, including squamous cell carcinoma, adenocarcinoma, large cell carcinoma, etc. There are certain differences in biological behavior an...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6886
CPCC12Q1/6886C12Q2600/154C12Q2600/166C12N15/11G01N35/00
Inventor 赵荣淞牛智通李仕良黄龙武吴幽治邹鸿志
Owner CREATIVE BIOSCIENCES (GUANGZHOU) CO LTD
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