Plasma free DNA methylation gene combination for identifying benign and malignant pulmonary nodules, and application of plasma free DNA methylation gene combination

A technology of gene combination and methylation, applied in the direction of recombinant DNA technology, DNA/RNA fragments, biochemical equipment and methods, etc., can solve the problems of difficult and invasive diagnosis of benign and malignant pulmonary nodules, and improve the detection accuracy , reduce the false positive rate, improve the detection of clinical sensitivity and clinical specificity

Pending Publication Date: 2020-12-18
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] The present invention aims to solve the problem of difficult and invasive clinical diagnosis of benign and malignant pulmonary nodules, and proposes a plasma free DNA methylation gene combination for differentiating benign and malignant pulmonary nodules.

Method used

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  • Plasma free DNA methylation gene combination for identifying benign and malignant pulmonary nodules, and application of plasma free DNA methylation gene combination
  • Plasma free DNA methylation gene combination for identifying benign and malignant pulmonary nodules, and application of plasma free DNA methylation gene combination
  • Plasma free DNA methylation gene combination for identifying benign and malignant pulmonary nodules, and application of plasma free DNA methylation gene combination

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0062] Example 1: Design of primer probe sets for 6 lung cancer-related genes

[0063] Query lung cancer-related genes SOX17, SCT, RASSF1A, SHOX2, HOXA7 and CDO1 and internal reference GAPDH through the National Center for Biotechnology Information (NCBI), and perform CpG through the Methyl Primer Express v1.0 software or "MethPrimer" launched by ABI. For island prediction, MethPrimer's default CpG island spans at least 200bp, GC content >50%, and CpG frequency >0.6. Therefore, regions that meet these parameters are defaulted to CpG islands. Find the 50 bp upstream and downstream of the position of the CpG island for sequence interception, and download and save the standard sulfurized sequence, that is, the cytosine except for the CpG dinucleotide is converted into uracil.

[0064] Design upstream and downstream primers and probes for the sequences of genes SOX17, SCT, RASSF1A, SHOX2, HOXA7, and CDO1 related to lung cancer and the internal reference GAPDH: use Methyl Primer Ex...

Embodiment 2

[0065] Example 2: Construction of the kit’s target positive reference product, internal reference gene GAPDH reference product, and construction of the target relative methylation standard linear equation obtained by transforming the target sequence plasmid into Escherichia coli, and SCT / SOX17 and 4 other lung malignancies The positive sequences after transformation of methylation-related genes and GAPDH detection targets are shown in Table 2. The plasmid pUC57 was inserted to construct a recombinant plasmid resistant to ampicillin. The recombinant plasmid was transformed into E. coli top10. The above part was handed over to Sangon Biological Engineering (Shanghai) Co., Ltd. for completion.

[0066] Table 2 Positive sequences after transformation of the detection target of the specific gene detected by the kit of the present invention

[0067]

[0068]

[0069] E. coli culture:

[0070] Escherichia coli in solid LB medium (tryptone 10g / L, yeast extract 5g / L, sodium chlo...

Embodiment 3

[0088] Example 3: Clinical sensitivity and clinical specificity verification of 6 different lung cancer methylation gene joint detection kits in detecting plasma cfDNA in patients with benign nodules and lung cancer patients

[0089] This example judges the gene combination selected in this kit by verifying the DNA methylation status of different combinations of 6 lung cancer-related methylation genes in plasma of lung cancer and the methylation status of plasma cell-free DNA in normal human plasma clinical sensitivity and clinical specificity.

[0090] Plasma cell-free DNA extraction:

[0091] use Serum / Plasma Circulating DNA Kit (purchased from Nanjing Novizan Biotechnology Co., Ltd., Cat. No. N902-01), was operated according to the kit instructions.

[0092] Plasma cell-free DNA bisulfite conversion:

[0093] The sulfite conversion was carried out according to the instruction manual of the EZ DNA Methylation-DirectTM KIT (D5031) of the ZYMO RESEARCH biological company k...

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Abstract

The invention discloses a plasma free DNA methylation gene combination for identifying benign and malignant pulmonary nodules, and application of the plasma free DNA methylation gene combination. Theplasma free DNA methylation gene combination for identifying benign and malignant pulmonary nodules is selected from at least any two of SOX17, SCT, RASSF1A, SHOX2, HOXA7 and CDO1. The invention discloses application of the plasma free DNA methylation gene combination as a detection target in preparation of a reagent for identifying benign and malignant pulmonary nodules. According to the technical scheme in the invention, a methylation relative quantification method is adopted; the GAPDH is mainly used as an internal reference; a methylation relative quantification reference product is constructed; and then, a relative methylation standard curve is made. The methylation degree of a lung malignant tumour related gene can be obtained through a standard curve; compared with methylation quantitative detection, the gene combination is more sensitive; the detection accuracy is improved; the false positive rate is reduced; and the gene combination has more clinical value for diagnosis of benign and malignant pulmonary nodules.

Description

technical field [0001] The invention belongs to the field of biological detection, and relates to a plasma free DNA methylation gene combination for differentiating benign and malignant pulmonary nodules and its application. Background technique [0002] In 2011, the National Lung Screening Trial (NLST) randomized controlled study showed that compared with chest X-rays, low-dose chest CT screening for high-risk groups can reduce the mortality rate of lung cancer by 20%. %, in view of the above research results, my country recommends that high-risk groups of lung cancer should undergo annual low-dose CT screening for early diagnosis of lung cancer. However, low-dose spiral CT can only identify pulmonary nodules, but its sensitivity to the differential diagnosis of benign and malignant pulmonary nodules is relatively low, that is, the false positive rate is as high as 96.4%, which brings some troubles to patients. According to the "Chinese Expert Consensus on the Diagnosis and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6886C12Q1/6883C12Q1/6851C12N15/11
CPCC12Q1/6886C12Q1/6883C12Q1/6851C12Q2600/154C12Q2600/112C12Q2600/166C12Q2531/113C12Q2561/101C12Q2523/125C12Q2545/101
Inventor 顾月清刘云龙胡子健
Owner CHINA PHARM UNIV
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