HOXA9 methylation detection reagent

A HOXA9, detection reagent technology, applied in the field of genetic diagnosis, can solve the problems of lack of non-invasive screening methods for lung cancer, difficult to meet the requirements of clinical use, and the sensitivity and specificity of tumor markers cannot meet the needs.

Active Publication Date: 2020-04-07
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
[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 ha

Method used

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  • HOXA9 methylation detection reagent
  • HOXA9 methylation detection reagent
  • HOXA9 methylation detection reagent

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0085] Example 1: Selection of Detection Target Genes

[0086] 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:

[0087] The det...

Embodiment 2

[0134] Example 2: Detection of HOXA9, SHOX2, PCDHGA12, HOXD8 and GATA3 genes in sputum

[0135] 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.

[0136] Experimental procedure:

[0137]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.

[0138] b. Use the DNA conversion kit (EZ DNA Methylation Kit, D5002) of ZYMO RESEARCH Biological Company to carry out the bisulfite modification of DNA.

[0139] c. The liquid distribution system is as...

Embodiment 3

[0150] Example 3: Detection of HOXA9 and SHOX2 genes in sputum

[0151] 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.

[0152] The detection primers and probes for each gene are as follows:

[0153] The detection primers and probes for HOXA9 are:

[0154] SEQ ID NO: 1HOXA9-F2 Primer ...

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Abstract

The invention relates to a lung cancer diagnostic reagent and a kit, and belongs to the field of genetic diagnosis. The reagent or the kit comprises a detection reagent aiming at HOXA9 gene methylation. The detection reagent or the kit provided by the invention has the advantages that the sensitivity in sputa is as high as 74.3 percent; the specificity is as high as 95 percent; the detection sensitivity is higher than that of an existing reported lung cancer tumor marker; the sensitivity in lavage fluid is as high as 61.9 percent; the specificity is as high as 95 percent; the sensitivity particularly on lung adenocarcinoma is greatly improved; and a huge application value is realized on the detection and diagnosis of the lung cancer.

Description

technical field [0001] The invention belongs to the field of gene diagnosis, and more specifically, the invention relates to a lung cancer diagnostic reagent based on human HOXA9 gene methylation or a kit containing the reagent. [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 and clinical course. According to the location of occurrence, it can be divided into: 1. Cen...

Claims

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Application Information

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IPC IPC(8): C12Q1/6886C12Q1/6858C12N15/11
CPCC12Q1/6886C12Q1/6858C12Q2600/154C12Q2523/125C12Q2531/113C12N15/11
Inventor 牛智通赵荣淞李仕良黄龙武吴幽治邹鸿志
Owner CREATIVE BIOSCIENCES (GUANGZHOU) CO LTD
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