Methylated molecular marker or combination thereof for detecting benign and malignant pulmonary nodules and application of methylated molecular marker or combination thereof

A technology of molecular markers and methylation, which is applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/testing, etc., to achieve the effect of improving patient survival rate, reducing false positive rate, and improving detection rate

Active Publication Date: 2021-11-12
ANCHORDX MEDICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] With the popularity of multi-slice spiral CT and high-resolution CT, and the improvement of people's health awareness, more and more pulmonary nodules have been detected, which is a good thing for patients, early lung cancer can be found, but it will also It causes a lot of trouble, and even many patients will feel uneasy in the bedroom after finding pulmonary nodules. Therefore, it is important to distinguish between benign and malignant pulmonary nodules in time.

Method used

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  • Methylated molecular marker or combination thereof for detecting benign and malignant pulmonary nodules and application of methylated molecular marker or combination thereof
  • Methylated molecular marker or combination thereof for detecting benign and malignant pulmonary nodules and application of methylated molecular marker or combination thereof
  • Methylated molecular marker or combination thereof for detecting benign and malignant pulmonary nodules and application of methylated molecular marker or combination thereof

Examples

Experimental program
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Effect test

Embodiment 1

[0155] A kit for the detection of benign and malignant pulmonary nodules in respiratory samples, including 13 detection genes B3GNTL1, DLX4, HOXA1, HOXA9, HOXB4, MSC-AS1, OTX2, PREX1, PTGER4, RASSF1, SHOX2, TWIST1 and ZNF781 The detection primers and probes of DNA methylation molecular markers Marker1 to Marker21 in 21 methylated regions, the detection region sequence and sequence number of each DNA methylation molecular marker are specifically shown in Table 1 (wherein each region The underlined part is the Marker of the corresponding sequence of the fragment amplified by the preferred primers used in the embodiment):

[0156] Table 1. Molecular marker detection region sequence

[0157]

[0158]

[0159]

[0160] The kit has designed three pairs of primers and three probes for each of the specific methylation sites in 21 molecular markers Marker1 to Marker21 for the detection of benign and malignant pulmonary nodules in respiratory samples (probe fluorescent labels c...

Embodiment 2

[0173] In this example, the kit described in Example 1 is used to detect the methylation levels of Marker1 to Marker21 in respiratory samples.

[0174] A method for detecting methylation levels of DNA methylation molecular markers, comprising the following steps:

[0175] 1. Extraction of gDNA from respiratory samples:

[0176] 1) Extraction of gDNA from paraffin section samples of lung tissue: the specific operation steps of gDNA extraction from paraffin tissue were carried out according to the instructions of Qiagen's ALLPrep DNA / RNA FFPE Kit;

[0177] 2) Extraction of gDNA from the respiratory fluid sample: First, the respiratory fluid sample was centrifuged at a low speed at 4°C, 5000g, for 5min; the supernatant was removed, and the precipitate was collected; Blood&Tissue Kit instructions were used to extract gDNA.

[0178] 2. Perform sulfite conversion on the extracted gDNA

[0179] The extracted gDNA is subjected to bisulfite conversion, and 20-50ng of gDNA is put in...

Embodiment 3

[0206] This embodiment provides the detection of molecular markers in standard products, and the detection steps are as follows:

[0207] 1. Standard product preparation

[0208] 1) Preparation of 0% methylation standard:

[0209] use Single Cell Kit (Qiagen, Cat#150343) and Mung Bean Nuclease (NEB, Cat#M0250L) were used to process NA12878 DNA to prepare 0% methylation standard;

[0210] 2) Preparation of 100% methylation standard:

[0211] The prepared 0% methylation standard was treated with CpG Methyltransferase (M.SssI) to obtain a 100% methylation standard.

[0212] 2. Preparation of standard products with different methylation ratios:

[0213] Mix 0% and 100% methylation standards according to the desired methylation ratio gradient to obtain 0.2%, 0.4%, and 1% methylation ratio standards.

[0214] 3. Perform bisulfite conversion on standard DNA with different methylation ratios: the steps are as in Example 2, and the conversion input amount is 10-50 ng, preferably ...

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Abstract

The invention provides a DNA (deoxyribonucleic acid) methylated molecular marker for detecting benign and malignant pulmonary nodules. The DNA methylated molecular marker is any one or a combination of more than two selected from sequences as shown in SEQ ID NO. 1 to SEQ ID NO. 21 or continuous fragments of at least 55% of the total length of the sequences as shown in SEQ ID NO. 1 to SEQ ID NO. 21; or is any one or a combination of more than two selected from complete complementary sequences of the sequences shown as SEQ ID NO. 1 to SEQ ID NO. 21 or continuous fragments of at least 55% of the full length of the complete complementary sequences of the sequences shown as SEQ ID NO. 1 to SEQ ID NO. 21. The invention also provides a detection kit and detection method of the DNA methylated molecular marker. The DNA methylated molecular marker is highly related to a lung cancer, and particularly when the DNA methylated molecular marker is used in combination, the sensitivity and specificity of detecting the benign and malignant pulmonary nodules can be further improved, the detection rate of malignant pulmonary nodules is increased, and the false positive rate of detection is reduced. Primers and probes in the kit overcome the defect that a plurality of primers and probes interfere with one another when multiple PCR amplification and detection are carried out, and the quantitative performance is equal to that of single-region quantification.

Description

technical field [0001] The invention belongs to the field of biotechnology, and specifically relates to a methylated molecular marker for detecting benign and malignant pulmonary nodules, its combination and application, and the application includes a detection kit and a detection method. Background technique [0002] Pulmonary nodules, that is, solitary pulmonary nodules (solitary pulmonary nodules) refer to images that are similar to circular shadows, single, well-defined, with a diameter of less than or equal to 3 cm, surrounded by high and low density of air-containing lung tissue Solid or subsolid lesions without atelectasis, hilar enlargement, or pleural effusion. [0003] Pulmonary nodules are divided into two types, benign and malignant, and often have no obvious symptoms. Benign nodules need to be treated according to the cause, while malignant nodules need early surgery. Benign etiologies are often associated with autoimmune diseases or various infections, and mal...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6883C12Q1/6851C12N15/11
CPCC12Q1/6886C12Q1/6883C12Q1/6851C12Q2600/112C12Q2600/154C12Q2600/16C12Q2531/113C12Q2537/143C12Q2563/107C12Q2523/125
Inventor 叶竹佳陶锦胜陈志伟范建兵
Owner ANCHORDX MEDICAL CO LTD
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