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DNA methylation qPCR kit for lung cancer detection and method of use
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A kit and methylation technology, applied in the fields of biotechnology and DN, can solve the problems of high sensitivity and selectivity of detection methods, poor DNA extraction methods, and poor bisulfite treatment capacity
Active Publication Date: 2021-09-14
NEOCURA BIO-MEDICAL TECH CO LTD
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Problems solved by technology
However, the main challenge of using ctDNA to assist cancer diagnosis and treatment selection in the clinic is how to develop a highly sensitive detection method to distinguish weak ctDNA signals from high cfDNA background levels, especially for ctDNA concentrations that may reach pg / mL levels. Early diagnosis requires higher sensitivity and selectivity of detection methods
Not only that, the current clinically available detection kits are generally limited by many limitations such as poor DNA extraction methods, poor bisulfite treatment, and unreasonable design of quantitative PCR methods for the detection of methylated DNA.
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Embodiment 1
[0078] Example 1: Using the kit of the present invention to perform multiple qPCR detection of lung cancer methylation on healthy human cfDNA samples
[0079] (1) Experimental materials
[0080] 1. Healthy human plasma (purchased from Bloodworks NW);
[0081] 2. QIAamp Circulatory System Nucleic Acid Extraction Kit (purchased from Qiagen, Cat. No. 55114);
[0082] 3. EZ DNA Methylation-Lightning Kit (purchased from Zymo Research, Cat. No. D5031);
[0083] 4. The kit of the present invention (including primers and probes for detection of target SHOX2, HOXA9 and TAC1, ACTB amplification primers and probes);
[0084] 5. AmpliTaq Gold TM DNA polymerase and buffer reagents (purchased from Thermo Fisher, Cat. No. 4311806).
[0085] (2) Experimental method
[0086] 1. DNA extraction from plasma
[0087] Cell-free DNA was extracted from 60 healthy human plasma samples using the QIAamp Circulatory System Nucleic Acid Extraction Kit, and the steps were performed according to the ...
Embodiment 2
[0101] Example 2: Using the kit of the present invention to perform multiple qPCR detection of lung cancer methylation on cfDNA samples from lung cancer patients
[0102] (1) Experimental materials
[0103] 1. Plasma from lung cancer patients (purchased from Bloodworks NW);
[0104] 2. QIAamp Circulatory System Nucleic Acid Extraction Kit (purchased from Qiagen, Cat. No. 55114);
[0105] 3. EZ DNAMethylation-Lightning Kit (purchased from Zymo Research, Cat. No. D5031);
[0106] 4. The kit of the present invention (including primers and probes for detection of target SHOX2, HOXA9 and TAC1, ACTB amplification primers and probes);
[0107] 5. AmpliTaq Gold TM DNA polymerase and buffer reagents (purchased from Thermo Fisher, Cat. No. 4311806).
[0108] (2) Experimental method
[0109] 1. DNA extraction from plasma
[0110] Cell-free DNA was extracted from 41 plasma samples of lung cancer patients using the QIAamp Circulatory System Nucleic Acid Extraction Kit, and the steps...
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Abstract
The invention belongs to the technical field of biotechnology and DNA detection, and in particular relates to a DNA methylation qPCR kit for lung cancer detection and its application method, in particular, to a methylation qPCR using plasma free DNA to determine The methylation status of one or more gene targets (such as SHOX2, HOXA9 and TAC1) is used for lung cancer detection or screening kits and methods of use thereof. After experimental testing, the kits of the present invention can combine the biomarkers The detection sensitivity is improved to the picogram / nanogram level of DNA molecules, and the detection sensitivity is improved by optimizing specific nucleotide sequence primers and DNA probes and improving the bisulfite treatment method of plasma free DNA.
Description
technical field [0001] The invention belongs to the technical field of biotechnology and DNA detection, and in particular relates to a DNA methylation qPCR kit for lung cancer detection and its application method, in particular, to a methylation qPCR using plasma free DNA to determine The methylation status of one or more gene targets (such as SHOX2, HOXA9 and TAC1) is used for the test kit for lung cancer detection or screening and the use method thereof. In addition, the present invention also relates to the application of the above detection kit in biomedicine Applications. Background technique [0002] Lung cancer is one of the leading causes of death worldwide. According to the American Cancer Society, there were approximately 234,030 new lung cancer cases and 154,050 lung cancer deaths in the United States alone in 2018, and the estimated number of lung cancer deaths is roughly equivalent to colon cancer, breast cancer, prostate cancer, leukemia, and non-Hodgkin lymph...
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