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Application of detection of methylation of lung cancer-related genes in the preparation of detection kits for lung cancer

A methylation and kit technology, applied in applications, genetic engineering, plant genetic improvement, etc., can solve the problems of high cost and complex technology of high-throughput sequencing, and achieve good application prospects, prolong survival, and sensitive specificity. Effect

Active Publication Date: 2021-06-29
FIRST HOSPITAL OF QINHUANGDAO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] At present, high-throughput sequencing has been used in the existing technology to prove that the methylation of hundreds of genes is related to the occurrence and development of lung cancer. However, high-throughput sequencing is expensive and technically complicated, and the existing lung cancer kits Most of them are single-gene tests, but the abnormally methylated DNA of the same gene only accounts for a very small part of the total DNA, about 0.1% to 1%. Abnormally methylated DNA detected in highly complex 'background'

Method used

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  • Application of detection of methylation of lung cancer-related genes in the preparation of detection kits for lung cancer
  • Application of detection of methylation of lung cancer-related genes in the preparation of detection kits for lung cancer
  • Application of detection of methylation of lung cancer-related genes in the preparation of detection kits for lung cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0019] Example 1 Specific primer design of the lung cancer related gene methylated markers:

[0020] According to LOC148898, VACM1 and RNS4 sequence information (NCBI Reference Sequence: NM_138479.2, NCBI Reference Sequence: NM_003478.3, NCBI Reference Sequence: NM_194430.1) open on the NCBI (the National Center for Biotechnology Information) website, using Primer Premier3 .0 and MethylPrimer Express V1.0 design, synthesized by Shanghai Introvigen Ltd..

[0021] The primer information synthesized is as follows:

[0022] Generation gene Upstream primer Downstream primer Loc148898 SEQ ID NO.1 SEQ ID NO.1 Vacm1 SEQ ID NO.3 SEQ ID NO.4 RNS4 SEQ ID NO.5 SEQ ID NO.6

Embodiment 2

[0023] Example 2 Joint test of LOC148898, VACM1 and RNS4 methylation levels in lung cancer samples:

[0024] Experimental materials: MASTER is purchased from Roche Ltd.; the DNA extraction kit is QIAMP DNA Mini Kit (Qiagen); the transformation kit is EZ DNA Methylation Kit (Zymo Research, Orange, CA, USA); other other Materials are all kinds of conventional market.

[0025] Experimental Sample: The 26 lung cancer tissue samples to be tested were from the first hospital of Qinhuangdao. USBIOLOGICAL.

[0026] experimental method:

[0027] 1. Take approximately 30 mg of lung cancer tissue or normal tissue to cut into 5 ml of centrifuge tube, and the DNA to be tested is extracted using a DNA extraction kit, and then the extraction DNA is converted by transforming the kit;

[0028] 2, PCR response procedure:

[0029]

[0030] Each of the components: Master 10UL, 25mm MgCl 2 3.2 ul, sterilization 5.8 ul, template 1 ul.

Embodiment 3

[0031] Example 3 Detection of other related gene methylation levels in lung cancer samples (ratio):

[0032] The pulmonary cancer height-associated gene HOXA9, PCDHGB6 known in the prior art will be combined with respect to the PCR reaction procedures and detection methods known in the prior art, and combined with the genes in Example 2. The primer sequences of the HOXA9 and PCDHGB6 are:

[0033] HOXA9:

[0034] Upstream primer: seq id number

[0035] Downstream primer: seq id No.:8

[0036] PCDHGB6:

[0037] Upstream primer: seq id No.:9

[0038] Downstream primer: seq id No.:10

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Abstract

The invention relates to a method and a kit for diagnosing the methylation of human LOC148898, VACM1 and RNS4. Specifically, it is to diagnose lung cancer and increase the detection rate of lung cancer by co-detecting the methylated DNA of three lung cancer markers, the lung cancer markers being human LOC148898, VACM1, and RNS4 genes. The kit of the invention can conveniently and quickly realize the screening of lung cancer at the molecular level, is non-invasive, has high detection rate and strong pertinence, and is beneficial to early detection and timely treatment of lung cancer.

Description

Technical field [0001] The present invention relates to molecular biological detection techniques, and in particular, to a polymeric methylation synergistic test kit associated with lung cancer. Background technique [0002] Lung cancer, also known as primary bronchial carcinoma, is one of the malignant tumors of human health, and the death rate is the first of various malignant tumors. In clinical practice, early diagnosis of lung cancer has always been difficult, but early detection is critical to effective treatment of cancer patients. At present, histological and cytological examination after clinical symptoms is a gold standard for lung cancer diagnosis, but early lung cancer is almost unaware of doctors and patients because there is no special symptoms, and it is difficult to detect early and early qualitative diagnosis by conventional diagnosis. And living sampling detection is also difficult, seriously affecting the early diagnosis of cancer and prognosis of patients. Sin...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6886C12Q1/6858C12N15/12C12N15/11
CPCC12Q1/6858C12Q1/6886C12Q2600/154C12Q2600/166C12Q2531/113C12Q2527/107C12Q2545/113
Inventor 高立明徐淑凤郑磊
Owner FIRST HOSPITAL OF QINHUANGDAO