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

A technology of molecular markers and methylation, applied in biochemical equipment and methods, recombinant DNA technology, microbial measurement/inspection, etc., can solve the problems of insufficient molecular detection of tissue samples, avoid excessive diagnosis and treatment, reduce False positive rate and the effect of improving the detection rate

Active Publication Date: 2022-06-21
ANCHORDX MEDICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Tissue samples are mainly obtained directly from the tumor site through surgery or fiberoptic bronchoscopic brushing, so the detection accuracy is higher. However, due to the invasiveness of tissue biopsy, the existence of tumor heterogeneity and various reasons, it cannot be obtained. Tissue samples or the amount of tissue samples are not enough to complete the molecular detection, so there are certain limitations in the role of tissue biopsy in the early diagnosis, prediction of metastasis and prognosis of lung cancer

Method used

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

Examples

Experimental program
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Effect test

Embodiment 1

[0151] A kit for the detection of benign and malignant pulmonary nodules in respiratory samples, including the DNA of 19 methylated regions in 10 detection genes CDO1, DAPK, GHSR, HOXA11, HOXB4, LHX9, MIR196A1, PTGER4, SHOX2 and TBX15 The detection primers and probes of the methylation molecular markers Marker1 to Marker19, the detection region sequence and sequence number of each DNA methylation molecular marker are specifically shown in Table 1 (the underlined part of each region is the following embodiment of the present invention Marker of the corresponding sequence of the fragment amplified by the preferred primers used):

[0152] Table 1. Molecular marker detection region sequence

[0153]

[0154]

[0155]

[0156] The kit has designed three pairs of primers and three probes for each of the specific methylation sites in the 19 molecular markers Marker1 to Marker19 for the detection of benign and malignant pulmonary nodules in respiratory samples (probe fluoresc...

Embodiment 2

[0169] In this example, the kit described in Example 1 is used to detect the methylation levels of Marker1 to Marker19 in respiratory samples.

[0170] The method for detecting the methylation level of the DNA methylation molecular marker comprises the following steps:

[0171] 1. Extraction of gDNA from respiratory samples:

[0172] 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;

[0173] 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.

[0174] 2. Perform sulfite conversion on the extracted gDNA

[0175] The extracted gDNA is subjected to bisulfite conversion, and 50-100ng of gDNA is...

Embodiment 3

[0191] This embodiment provides a detection method for molecular markers in standard products, and the detection steps are as follows:

[0192] 1. Standard product preparation

[0193] 1) Preparation of 0% methylation standard:

[0194] 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;

[0195] 2) Preparation of 100% methylation standard:

[0196] The prepared 0% methylation standard was treated with CpG Methyltransferase (M.SssI) to obtain a 100% methylation standard.

[0197] 2. Preparation of standard products with different methylation ratios:

[0198] Mix 0% and 100% methylation standards according to the desired methylation ratio gradient to obtain 0.2%, 0.4%, and 1% methylation ratio standards.

[0199] 3. Perform bisulfite conversion on the standard DNA with different methylation ratios: the steps are as in Example 2, and the conversion input amount is 50-100 ng, ...

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Abstract

The invention provides a DNA methylation molecular marker or a combination thereof for detecting benign and malignant pulmonary nodules. The DNA methylation molecular marker or the combination thereof is selected from any one or a combination of more than two of sequences as shown in SEQ ID NO.1-SEQ ID NO.19 or continuous fragments of at least 55% of the full length of the sequences as shown in SEQ ID NO.1-SEQ ID NO.19; 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-SEQ ID NO.19 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-SEQ ID NO.19. Wherein in particular, the SEQ ID NO.9 has relatively high specificity for detecting benign and malignant pulmonary nodules. The invention also provides a detection kit and a detection method of the DNA methylation molecular marker. The DNA methylation molecular marker is highly related to lung cancer, and especially when the DNA methylation molecular marker is used in combination, the sensitivity and specificity of detecting 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. The primer and the probe in the kit overcome the defect that a plurality of primers and probes interfere with each other when multiple PCR amplification and detection are carried out, and the quantitative performance is equal to that of single-region quantification.

Description

[0001] The present invention claims the priority of the Chinese patent application CN2020114961847 entitled "Methylated Molecular Markers for Detecting Benign and Malignant Pulmonary Nodules and Its Combination and Application" filed on December 17, 2020, and the entire description and disclosure of the invention Incorporated here by reference; the present invention also requires the title of the PCT application of PCT / CN2021 / 086902 filed on April 13, 2021 entitled "Methylated Molecular Markers for Detecting Benign and Malignant Pulmonary Nodules and Their Combinations and Applications" Priority, the disclosure of this invention regarding the methylation molecular marker combination M, N, O is hereby incorporated by reference. technical field [0002] 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 d...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/6886C12Q1/6851C12N15/11
CPCC12Q1/6883C12Q1/6886C12Q1/6851C12Q2600/154C12Q2600/166C12Q2600/16C12Q2563/107C12Q2531/113C12Q2545/101C12Q2523/125C12Q2537/143C12N15/11
Inventor 叶竹佳杨昊陈思宇陈志伟范建兵
Owner ANCHORDX MEDICAL CO LTD
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