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Kit for identifying lung nodules and/or lung cancer status and application thereof

A kit and technology for pulmonary nodules, applied in the field of kits for identifying pulmonary nodules and/or lung cancer status, to achieve the effects of improving sensitivity and specificity, rapid diagnosis and evaluation, and ensuring correctness and reliability

Inactive Publication Date: 2018-12-18
BEIJING EXELLON MEDICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is still a lack of means to effectively detect the methylation status of these cancer-related genes and process the detection results

Method used

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  • Kit for identifying lung nodules and/or lung cancer status and application thereof
  • Kit for identifying lung nodules and/or lung cancer status and application thereof
  • Kit for identifying lung nodules and/or lung cancer status and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0061] Example 1: DNA Extraction

[0062] DNA Extraction Reagent consists of Lysis Buffer, Binding Buffer, Washing Buffer and Elution Buffer. Lysis buffer consists of protein denaturants, detergents, pH buffers and nuclease inhibitors. Binding buffers consist of protein denaturants and pH buffers. The cleaning buffer is divided into cleaning buffer A and cleaning buffer B, and cleaning buffer A is composed of protein denaturant, nuclease inhibitor, detergent, pH buffer and ethanol; cleaning buffer B is composed of nuclease inhibitor, pH buffer and ethanol composition. The elution buffer consists of nuclease inhibitors and pH buffers. The protein denaturant is: guanidine hydrochloride; the detergent is: Tween20; the pH buffer is: Tris-HCl; the nuclease inhibitor is: EDTA.

[0063] In this embodiment, the plasma samples of lung cancer patients are taken as an example to extract plasma DNA. The extraction method comprises the following steps:

[0064] (1) Take 1ml of plasma...

Embodiment 2

[0076] Example 2: Bisulfite Treatment of DNA

[0077] Bisulfite treatment of DNA is to use bisulfite reagent to treat the extracted DNA sample. The bisulfite reagent is composed of bisulfite buffer and protection buffer; the bisulfite buffer is bisulfite A mixture of sodium and water; the protection buffer is a mixture of hydroquinone, an oxygen free radical scavenger, and water.

[0078] In this embodiment, the DNA extracted in Example 1 is used as the processing object, and the DNA is treated with bisulfite, and the specific steps include:

[0079] (1) Preparation of bisulfite buffer: weigh 1g of sodium bisulfite powder, add water to prepare 3M buffer;

[0080] (2) Preparation of protection buffer: weigh 1g of hydroquinone reagent, add water to prepare 0.5M protection buffer;

[0081] (3) Mix 100 μl DNA solution, 200 μl bisulfite buffer solution and 50 μl protection solution, shake and mix evenly;

[0082] (4) Thermal cycle: 95°C for 5 minutes, 80°C for 60 minutes, 4°C fo...

Embodiment 3

[0093] Example 3: Detection of DNA methylation by real-time fluorescent PCR and verification of primer sets

[0094] In this embodiment, real-time fluorescent PCR is used as an example to detect the methylation level of biomarker genes. The detected genes were APC, DAPK, FHIT, FOXL2, HOXA9, MGMT, P16, PTGER4, RASSF1A, SHOX2, SOX17 and ZAR1 genes, and the internal reference gene was ACTB. In this example, the DNA treated with bisulfite in Example 2 was used as a template for real-time fluorescent PCR amplification. The DNA samples to be tested, the negative quality control, the positive quality control, and the no-template control were all tested in triplicate holes.

[0095] For APC, DAPK, FHIT, FOXL2, HOXA9, MGMT, P16, PTGER4, RASSF1A, SHOX2, SOX17 and ZAR1 genes, many sets of primer and probe combinations can be designed, and the performance of each set of probe-primer combinations may vary. difference, so it needs to be verified by experiments. In this example, the prime...

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Abstract

The present invention relates to a kit for identifying lung nodules and / or lung cancer status and application thereof. The kit includes a primer pair for detecting methylation levels of biomarker genes or corresponding fragments in a biological sample from a subject; the primer pair is used for PCR amplification by use of the biomarker genes or the fragments treated with hydrosulphite as templates; and the biomarker genes are selected from one or more of APC, DAPK, FHIT, FOXL2, HOXA9, MGMT, P16, PTGER4, RASSF1A, SHOX2, SOX17 and ZAR1. The method improves the detection sensitivity and specificity of the lung nodules and lung cancer by jointly detecting the methylation levels of the biomarker genes or the corresponding fragments, thus ensuring the correctness and reliability of detection results, and provides a fast, reliable and accurate new way for the prediction, diagnosis and evaluation of the lung nodules and the lung cancer.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a kit for identifying the status of pulmonary nodules and / or lung cancer and its application. Background technique [0002] Lung cancer is a malignant tumor with the highest morbidity and mortality rate in the world, and it is a major disease that threatens human life and health. The 2014 World Cancer Report released by the International Agency for Research on Cancer (IARC) of the World Health Organization shows that the global cancer burden is increasing, and lung cancer ranks first among the new common cancers in 2012, accounting for about 1.8 million cases, accounting for 20% of the total number of common cancers. 13%; in the common death cancer, lung cancer also ranks first, about 1.6 million cases, accounting for 19.4% of the total. Among the top ten cancers with high incidence, lung cancer has the highest incidence in men, accounting for 23%, and 14.85% in women, second only t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12Q1/6886
CPCC12Q1/6883C12Q1/6886C12Q2600/154C12Q2600/166
Inventor 陈彦利蒲珏李明明李淑钰徐春叶
Owner BEIJING EXELLON MEDICAL TECH CO LTD
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