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Application of methylation of lung carcinoma detection related gene to preparation of lung carcinoma detection kit

A technology of methylation and kits, which is applied in the fields of application, genetic engineering, plant gene improvement, etc., can solve the problems of complex technology and high cost of high-throughput sequencing, etc., and achieve prolonged survival, good application prospects, sensitivity and specificity Effect

Active Publication Date: 2020-11-06
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'

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  • Application of methylation of lung carcinoma detection related gene to preparation of lung carcinoma detection kit
  • Application of methylation of lung carcinoma detection related gene to preparation of lung carcinoma detection kit
  • Application of methylation of lung carcinoma detection related gene to preparation of lung carcinoma detection kit

Examples

Experimental program
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Effect test

Embodiment 1

[0019] Example 1 Design of specific primers for methylation markers of lung cancer-related genes:

[0020] According to the LOC148898, VACM1 and RNS4 sequence information (NCBI Reference Sequence: NM_138479.2, NCBI Reference Sequence: NM_003478.3, NCBI Reference Sequence: NM_194430.1) published on the website of NCBI (National Center for Biotechnology Information), use Primer Premier3 .0 and MethylPrimer Express v1.0 design, synthesized by Shanghai Introvigen Co., Ltd.

[0021] The synthesized primer information is as follows:

[0022] gene to be tested 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 detection of LOC148898, VACM1 and RNS4 methylation levels in lung cancer samples:

[0024] Experimental materials: Master was purchased from Roche Co., Ltd.; DNA extraction kit was QIAmp DNA Mini Kit (QIAGEN); transformation kit was EZ DNA Methylation kit (Zymo Research, Orange, CA, USA); other materials used in the experiment The materials are all conventional commercially available species.

[0025] Experimental samples: The 26 lung cancer tissue samples to be tested were all from the First Hospital of Qinhuangdao City. Human lung cancer cell lines A549, PC-9, SPCA-1 and SK-MES-1 were used as positive controls. Normal adult chromosomal DNA was purchased from USB Biological.

[0026] experimental method:

[0027] 1. Take about 30mg of lung cancer tissue or normal tissue, cut it into pieces and add it to a 5ml centrifuge tube, use the DNA extraction kit to extract the DNA of the sample to be tested, and then use the transformation kit to transform the ...

Embodiment 3

[0031] Example 3 Detection of methylation levels of other related genes in lung cancer samples (comparative example):

[0032] Referring to the PCR reaction program and detection method in Example 2, the known high lung cancer related genes HOXA9 and PCDHGB6 were detected in the prior art and combined with the genes in Example 2 respectively. The primer sequences of HOXA9 and PCDHGB6 are respectively:

[0033] HOXA9:

[0034] Upstream primer: SEQ ID NO.:7

[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 kit for diagnosing human LOC148898, VACM1 and RNS4 methylation. Particularly, the lung carcinoma is diagnosed and the detection rate of lung carcinoma is increased by synergistically detecting methylated DNAs of three lung carcinoma markers; and the lung carcinoma markers are human LOC148898, VACM1 and RNS4 genes. The kit of the invention can conveniently andquickly realize screening of the lung carcinoma at a molecular level, is non-invasive, has a high detection rate and is highly targeted, and is beneficial to the early detection and timely treatment of the lung carcinoma.

Description

technical field [0001] The invention relates to molecular biological detection technology, in particular to a multi-gene methylation cooperative detection kit related to lung cancer. Background technique [0002] Lung cancer, also known as primary bronchial cancer, is one of the most harmful malignant tumors to human health, and its mortality rate ranks first among all kinds of malignant tumors. In clinical practice, early diagnosis of lung cancer has always been difficult, but early detection is crucial to the effective treatment of cancer patients. At present, histological and cytological examinations after the appearance of clinical symptoms are the gold standard for the diagnosis of lung cancer. However, early lung cancer is hardly noticed by doctors and patients because it often has no special symptoms, and it is difficult to detect and diagnose early with conventional diagnostic methods. , and the detection of live samples is also difficult, which seriously affects th...

Claims

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Application Information

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Patent Type & Authority Applications(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